In eukaryotic cells, DNA replication starts at many origins in each chromosome during S phase of cell cycle. Each origin is activated at different time points in S phase, which takes place once and only once per cell cycle. In yeast and most likely higher eukaryotes, the origin-recognition complex (ORC) and several other initiation factors play a pivotal role in activation and regulation of replication origins. Briefly, the ORC-bound origins are sequentially activated and deactivated along the progression of cell cycle. The prereplicative complex (pre-RC) is formed by loading the replicative helicase MCM complex onto the ORC-bound origins with the aid of Cdc6 and Cdt1. This complex is activated by S-phase cyclin dependent kinases (Cdks) when cells enter S phase. The elevated levels of Cdk activities lead to removal of some initiation proteins such as Cdc6 by proteolysis, allowing the pre-RC to be further activated for subsequent DNA synthesis. The irreversible removal of initiation factors is a major mechanism to ensure DNA to be replicated once and only once per cell cycle. The assembly of replication initiation complex and its activation are well reviewed in many literatures (Sclafani & Holzen, 2007; Remus & Diffley, 2009; Araki, 2010). Activation of origins leads to the establishment of bidirectional replication forks for the DNA synthesis of leading and lagging strands.
2. Overview of lagging strand synthesis
Leading strand synthesis, once initiated, occurs in a highly processive and continuous manner by a proofreading DNA polymerase. Unlike leading strands, lagging strands are synthesized as discrete short DNA fragments, termed ‘Okazaki fragments’ which are later joined to form continuous duplex DNA. Synthesis of an Okazaki fragment begins with a primer RNA-DNA made by polymerase (Pol) α-primase. The synthesis of RNA portion (~ 10 to 15 ribonucleotides) and subsequent extension of short (~20 to 30 nucleotides, nt) DNA are coupled. The recognition of a primer RNA-DNA by the Replication-Factor C (RFC) complex leads to dissociation of Pol α-primase and loading of proliferating cell nuclear antigen (PCNA), resulting in recruitment of Pol δ to the primer-template junction, a process called ‘polymerase switching.’ Then the primer RNA-DNA is elongated by Pol δ. When Pol δ encounters a downstream Okazaki fragment, it displaces the 5’ end region of the Okazaki fragment, generating a single-stranded (ss) nucleic acid flap. The flaps formed can be efficiently processed by the combined action of Flap endonuclease 1 (Fen1) and Dna2 to eventually create nicks. The nicks are finally sealed by DNA ligase 1 to complete Okazaki fragment processing. The current model is summarized in Fig. 1.
3. Potential risks associated with lagging strand synthesis in eukaryotes
Lagging strand maturation appears to be intrinsically at high risks of suffering DNA alterations for several reasons. First, a substantial part (up to 20%) of short Okazaki fragments (~150-nt in average) is synthesized by Pol α which does not contain a proofreading function (Conaway and Lehman, 1982; Bullock et al., 1991). Thus, the high-incidence errors in Okazaki fragments, if not effectively removed, could become a source of genome instability. Second, the
4. ‘Core’ factors for synthesis and maturation of lagging strands
The protein factors required for synthesis of lagging strands include Pol α-primase, Pol δ, PCNA, RFC, RPA, Fen1 (5’ to 3’ exonuclease or MF1, maturation factor 1), RNase H, and DNA ligase 1. In essence, a combined action of these factors was sufficient and necessary for completion of lagging strand synthesis in vitro in simian virus 40 DNA replication (Ishimi et al., 1988; Waga & Stillman, 1994). Among them, the two nucleases Fen1 and RNase H were shown to have roles in the removal of primer RNA of Okazaki fragments. In yeasts, however, the deletion of genes encoding Fen1 (RAD27) or RNase H (RNH35) was not lethal, indicating the presence of redundant pathways in eukaryotes (Tishkoff et al., 1997a; Qiu et al, 1999). In addition, Dna2, which was originally reported as a helicase (Budd & Campbell 1995; Budd et al., 1995), was shown to play a critical role in the processing of Okazaki fragments using its endonuclease activity (Bae et al., 1998; Bae & Seo, 2000; Bae et al., 2001a; MacNeill, 2001; Kang et al., 2010). Displacement DNA synthesis by Pol δ generates flap structures, which can be substrates for Dna2 and Fen1 endonuclease activities (Bae & Seo, 2000). For the convenience sake, all enzymes (Pol δ, PCNA, RFC, RPA, Fen1, RNase H, Dna2, and DNA ligase 1) described early from yeast and human studies are referred to as ‘core’ factors for synthesis of lagging strands in this chapter. We refer to all the others as ‘auxiliary’ factors which may not be needed normally, but become critical under specific circumstances (Fig. 1 and see also Fig. 3). These factors have been screened for their abilities to suppress the crippled function of Dna2 or Fen1. It is believed that (i) the ‘auxiliary’ factors come to assist the ‘core’ machinery that does not function appropriately, (ii) they provide additional enzymatic activities to resolve hairpin or higher-ordered structures in flaps, or (iii) they are needed to resolve toxic recombination intermediates arising during lagging strand metabolism. Thus, it is the multiplicity of ‘auxiliary’ factors that allows the ‘core’ machinery to be fine-tuned in response to diverse situations with regard to Okazaki fragment processing.
4.1. Multiple pathways in parallel with Fen1
Fen1 is a major, but not the only enzyme that can create ligatable nicks directly from flap structures (Harrington & Lieber, 1994; Murante et al., 1995; Liu et al., 2004; Garg & Burgers 2005). In vivo studies demonstrated that double-strand break(DSB)-induced DNA repair, which requires replication of both leading and lagging strands, still occurred 50% in Fen1-deficient strains compared to wild type (Holmes & Haber, 1999), indicating that the 50% of the repair events were carried out with nicks created by nuclease(s) other than Fen1. The ability of Pol δ to switch from displacement DNA synthesis to its 3’ exonuclease could constitute a pathway to create nicks; the retrograde 3’ exonucleolytic degradation of a newly elongated end, followed by annealing of the displaced flap to the lagging strand template, can be a mechanism for nick formation (Jin et al., 2001). The overexpression of Exo1 in rad27 restored growth of the mutant cells at the nonpermissive temperature (Tishkoff et al., 1997b). Single mutant cells with either rad27 or exo1 were viable, whereas rad27( exo1( double mutants were not (Budd et al., 2000; Tishkoff et al., 1997b). Yeast Exo1 has 5′ exonuclease activity acting on double stranded (ds) DNA and an associated 5′-flap endonuclease activity (Tran et al., 2001). In addition, yeast rad27Δ cells (lacking yeast Fen1) were not lethal, but temperature-sensitive (ts) in growth, consistent with existence of multiple pathways for nick generation in yeasts. It was shown that Pol δ has a unique ability to maintain dynamically the nick position in conjunction with Fen1, via a process called ‘idling’. In addition, Pol δ cooperates with Fen1 and PCNA to carry out ‘nick translation’ to progressively remove primer RNA-DNAs (Garg et al., 2004). The endonuclease activity of Fen1 can keep cleaving a flap while it is being displaced by Pol δ, allowing nicks to be changed in their positions along with Pol δ movement.
4.2. Structured flaps are special types of DNA damage that could cause genome instability
Failure to create nicks by Fen1 in a timely manner could cause genome instability. The importance of Fen1 in this regard was clearly demonstrated by the dramatic increase of small (5- to 108-bp) duplications flanked by 3- to 12-bp repeats in rad27Δ mutants (Tishkoff et al., 1997a). This unusual type of duplication mutations is in keeping with the current model of Okazaki fragment processing; unprocessed flaps, rapidly accumulated in the absence of Fen1, are ligated with the 3′-end of the downstream Okazaki fragment, resulting in duplication mutations. In the absence of Fen1, many types of repeat DNA sequences in eukaryotic chromosomes are not stably maintained. These include dinucleotide, trinucleotide, micro- or mini-satellite DNA, and telomeric DNA (Johnson et al., 1995; Kokoska et al., 1998; Xie et al., 2001; Freudenreich et al., 1998; Spiro et al., 1999; White et al., 1999; Maleki et al., 2002; Lopes et al., 2002; Lopes et al., 2006). Most notably, expansion of trinucleotide repeats such as CTG/CAG or CGG/CCG has been extensively studied using yeasts as model system (Schweitzer & Livingston, 1998; Freudenreich et al., 1998; Shen et al., 2005), because of their clinical relevance to many human neurodegenerative diseases such as Fragile X Syndrome, Huntington’s Disease, and Myotonic Dystrophy (Pearson et al., 2005; Kovtun & McMurray, 2008). All of the disease-causing trinucleotide repeats are able to form secondary or higher-ordered structures in solution, such as hairpins (CAG, CTG, CGG, and CCG repeats), G quartets (CGG repeats), and triplexes (GAA and CTT) (Fig. 2).
Trinucleotide repeats, once displaced by Pol δ, could reanneal to the template in a misaligned manner. If they are joined to the 3’ end of the new Okazaki fragment, followed by a subsequent round of DNA replication, the repeats could be expanded. In yeast, stability of trinucleotide repeats is greatly affected by their orientation with respect to nearby replication origins (Freudenreich et al., 1997; Miret et al., 1998). The orientation-dependent and sequence-specific instability of trinucleotide repeats support the model that expansions of CTG and CAG tracts result from aberrant DNA replication via hairpin-containing Okazaki fragments. In addition, telomere repeats are not stably maintained in the absence of functional Fen1 in yeasts (Parenteau & Wellinger, 1999 and 2002). Although Fen1 is critical for repeat stability in yeasts, it remains unclear in mice or humans (Spiro & McMurray, 2003; Moe et al., 2008; van den Broek et al., 2006). One explanation is that unlike yeasts, mammals may have more diverse pathways to remove or prevent formation of long flaps, since instability of the trinucleotide repeats occurs through formation of long flaps. Alternatively, Fen1 is responsible for formation of most nicks in mammals because deletion of Fen1 caused embryonic lethality in mice (Kucherlapati et al., 2002). The human minisatellite DNA became unstable in rad27 or dna2 mutant cells when it was inserted into one of the yeast chromosomes (Lopes et al., 2002; Cederberg & Rannug, 2006). These data also are in keeping with the idea that improperly processed 5’ flap instigates minisatellite destabilization. DNA instability associated with secondary or higher-ordered structures in the flap indicates that structures formed during DNA metabolisms can be regarded as special forms of DNA damage that need to be immediately removed (Fig. 2). The role of Fen1 in safeguarding the genome integrity has qualified Fen1 as a tumor suppressor in mammals and its physiological importance was recently reviewed with an emphasis on studies of human mutations and mouse models (Zheng et al., 2011).
4.3. Dna2 as a preemptive means to prevent formation of long flaps
4.3.1. Long flaps are in vivo substrates preferred by Dna2
Dna2 is highly conserved throughout eukaryotes and contains at least two catalytic domains for helicase and endonuclease activities (Budd & Campbell, 1995; Budd et al., 1995; Bae et al., 1998; Bae et al., 2001b). Genetic data from fission and budding yeasts indicate that the endonuclease activity of Dna2 is essential, playing an essential role in vivo in Okazaki fragment processing (Kang et al., 2000; Lee et al., 2000; Budd et al., 2000; Kang et al., 2010). There are several lines of evidence that long flaps can be formed in vivo that need the action of Dna2. Long flaps, once formed, could impose formidable burdens to cells, most likely due to their tendency to bind proteins nonspecifically or to form hairpin or higher-ordered structure that is difficult to be processed. In this sense, any structural intermediates formed in flaps can be regarded as a special type of DNA damage. The requirement of Dna2 endonuclease and helicase activities for a complete removal of long or hairpin flaps supports the idea that the major role of Dna2 is to prevent formation of excessively long flaps by cleaving them into shorter ones as soon as they occur. The flaps shortened by Dna2 are not able to form secondary or higher-ordered structure. Thus, Dna2 functions to maintain flaps as short as possible during replication. The marked increase of unusual duplications or trinucleotide expansions in the absence of Fen1 (Tishkoff et al., 1997a) provide strong evidence that long flaps are produced in vivo. It was shown that calf thymus Pol δ was able to displace downstream duplex DNA longer than 200 bps in vitro, revealing its intrinsic ability to form extensive flaps (Podust & Hubscher, 1993; Podust et al., 1995; Maga et al., 2001). In vitro reconstitution experiments using yeast enzymes showed that a portion of flaps grows long up to 20- to 30-nt, although flaps formed in vitro are primarily short, up to 8-nt in length (Rossi & Bambara, 2006). The frequency of long flaps can be affected by sequence in the lagging strand template or by interactions of Pol δ/Dna2 with other proteins. For example, Pol δ lacking PCNA-interaction tends to preferentially generate short flaps (Jin et al., 2003; Garg et al., 2004; Tanaka et al., 2004). In contrast, Pif1 helps to create long flaps through its helicase activity in vitro (Rossi et al., 2008) and in vivo (Ryu et al., 2004). Several other elaborate genetic experiments are in keeping with involvement of Dna2 in the cleavage of long flaps. First, dna2-1 was lethal in combination with a mutation in Pol δ (pol3-01) which increased strand displacement synthesis. Meanwhile, deletions of Pol32 subunit, which reduces strand displacement activity of Pol δ in vitro, suppressed the growth defects of dna2-1 and dna2-2 (Burgers & Gerik, 1998; Garg et al., 2004; Johansson et al., 2004). Similar results were also obtained in S. pombe (Reynolds et al., 2000; Zuo et al., 2000; Tanaka et al., 2004). The observation that overexpression of RPA alleviates the requirement of Dna2 helicase activity (Bae et al., 2002) is also consistent with formation of long flaps in vivo. In order for dsDNA-destabilizing activity of RPA to substitute for the helicase activity of Dna2, flaps should be at least long enough to form hairpin structure.
4.3.2. RPA acts as a molecular switch between Dna2 and Fen1
Several independent observations indicate that RPA plays a critical role in Okazaki fragment processing in conjunction with Dna2; (i) a mutation in DNA2 was identified during a synthetic lethal screen with rfa1Y29H, a ts mutant allele of RFA1. Furthermore, Dna2 and Rpa1 (a large subunit of RPA encoded by RFA1) physically interacted with each other both in vivo and in vitro (Bae et al., 2003). (ii) The 32 kDa subunit of RPA was crosslinked to primer RNA–DNA in the lagging strand of replicating SV40 chromosomes (Mass et al., 1998). (iii) The genetic interaction between RPA and Dna2 was discovered from screening of suppressors that rescued ts growth defects of dna2Δ405N mutant when expressed in a multicopy plasmid (Bae et al., 2001a). The fact that RPA binds most efficiently ssDNA longer than 20-nt and interacts genetically with Dna2 is consistent with the idea that the in vivo substrates of Dna2 are long ssDNA flaps. In vitro, RPA markedly stimulated Dna2-catalyzed cleavage of 5’ flap at physiological salt concentration (Bae et al., 2001a), which was further confirmed by others (Ayyagari et al., 2003; Kao et al., 2004). However, RPA inhibited Fen1-catalyzed cleavage of 5’ flaps. This inhibition was readily relieved by the addition of Dna2 (Bae et al., 2001a). Thus, a 5’ flap longer than 20-nt first binds RPA, and then rapidly recruits Dna2 to form a ternary complex. Dna2-catalyzed cleavage of the flap releases free RPA-bound ssDNA and a shortened flap (mostly 6-nt). The short flap produced is no longer resistant to and can be completely removed by Fen1 to produce ligatable nicks. Therefore, RPA acts as a molecular switch between Dna2 and Fen1 for the sequential action in cleavage of long flaps, Dna2 followed by Fen1, of the two endonucleases (Bae et al., 2001a).
4.3.3. A concerted action of helicase and endonuclease activities for removal of hairpin flaps
The presence of both endonuclease and helicase activities in one polypeptide of Dna2 implies that both activities act in a collaborative manner. The lethality of dna2 mutation lacking helicase activity (Budd et al., 1995) suggests that DNA unwinding activity is critical for its physiological function in vivo. The addition of ATP not only activates helicase activity, but also alters the cleavage pattern of flap DNA by Dna2. The average size of cleaved flaps is expanded in the presence of ATP (Bae et al., 2002). Furthermore, the addition of ATP allowed wild type Dna2, not helicase-negative Dna2K1080E mutant, to cleave secondary-structured flap via its combined action of helicase and nuclease activities (Bae et al., 2002). The mixture of helicase-negative Dna2K1080E and nuclease-negative Dna2D657A mutant enzymes failed to recover wild type action on these structured flaps. Therefore, it is critical essential that these two essential activities should be concerted. In keeping with this, simultaneous expression of both mutant proteins in dna2Δ cell did not allow cells to grow. Dna2 is also capable of unwinding G-quadruplex DNA structures, suggesting another critical role of Dna2 helicase in resolving the structural intermediates arising during DNA metabolisms (Masuda-Sasa et al., 2008). It was also shown that concerted action of exonuclease and gap-dependent endonuclease activities of Fen1 could contribute to the resolution of trinucleotide-derived secondary structures formed during maturation of Okazaki fragments (Singh et al., 2007).
4.3.4. Dna2 as an alternative means to remove mismatches
Since the Pol α-synthesized DNA in Okazaki fragments is highly mutagenic, eukaryotic cells need to eliminate this mutagenic DNA to prevent accumulation of errors. Recently, it was shown that in yeast Pol α incorporates ribonucleotides more frequently than Pol δ or Pol ε (Nick McElhinny et al., 2010b). The unrepaired ribonucleotides in DNA could inflict a potential problem on DNA replication because Pol ε has difficulty bypassing a single ribonucleotide present within a DNA template in yeasts. This again emphasizes that processing of Okazaki fragments is associated with high risks of DNA alterations. It has been puzzling that eukaryotic cells maintain a low mutation rate, despite the fact that a substantial portion (~10%) of total DNA is synthesized by Pol α, a flawed DNA polymerase. To account for this enigma, it was proposed that in mammals Pol α is associated with a 3’ exonuclease that may confer a proofreading function on Pol α (Bialek and Grosse, 1993). In yeasts, an intermolecular proofreading mechanism was proposed in which Pol δ could play a role in proofreading errors made by Pol α during initiation of Okazaki fragments (Pavlov et al., 2006). Mismatch repair (MMR) can correct mismatches in the Pol α-synthesized DNA (Modrich & Lahue 1996; Kolodner & Marsischky,1999; Kunkel & Erie, 2005). One unsolved fundamental problem in eukaryotic MMR, however, is the strand discrimination signal, although a strand-specific nick is generally believed to be the signal (Holmes et al., 1990; Thomas et al., 1991; Modrich, 1997). Equally possible is that the presence of flaps, which may be as abundant as nicks in lagging strand, could act as the strand discrimination signal. At any rate, the accuracy of MMR would depend on the rate at which nicks or flaps (the strand discrimination signals) are being removed. Thus, MMR could be unreliable if MMR is kinetically slower than sealing nicks. The ability of Dna2 to efficiently remove the RPA-bound flap containing the whole RNA-DNA primer could offer an alternative mechanism to remove mismatches present in the primer DNA of Okazaki fragments.
5. Multi-factorial interplays as a means to ensure high-fidelity replication of lagging strand
If one of the ‘core’ factors is crippled, a redundant factor(s) that works in parallel can reveal itself. In our laboratory, we have focused on isolating genetic suppressors that can rescue dna2 mutations in order to identify redundant pathways for Okazaki fragment processing. Most suppressors isolated turned out to have roles in maintenance of genome integrity, in keeping with the notion that faulty processing of Okazaki fragment could lead to genome instability. The in vivo and in vitro interactions of the suppressors with Dna2 or Fen1 suggest that Okazaki fragment processing is a converging place for DNA replication, repair, and recombination proteins to ensure removal of flaps in an accurate and timely manner in eukaryotes.
5.1. RNase H2 as an enzyme to clean up ribonucleotides in lagging strands
Both type I and type II RNase H play a role in the removal of ribonucleotides present in duplex DNA (Ohtani et al., 1999; Cerritelli & Crouch, 2009). The S. cerevisiae RNase H2 enzyme is active as a heterotrimeric complex that consists of Rnh201, Rnh202, and Rnh203, which are encoded by RNH201 (formerly known as RNH35), RNH202, and RNH203, respectively (Jeong et al., 2004). Expression analyses and other results suggest that RNase H2 plays roles in DNA replication and/or repair (Frank et al., 1998; Qiu et al., 1999; Arudchandran et al., 2000). Since rnh201Δ and rnh202Δ displayed synthetic lethal interactions with dna2-1 and rad27Δ, yeast RNase H2 has been implicated in Okazaki fragment processing (Budd et al., 2005). The unique ability of eukaryotic RNase H2 (type II) to cleave the 5’ side of a single ribonucleotide embedded within duplex DNA suggests an additional role, that is, the removal of ribonucleotides misincorporated into DNA (Rydberg & Game, 2002). The catalytic activity of RNase H2 was critical for a pathway requiring the function of RAD27 since all rnh201 mutant alleles failed to complement the growth defect of rad27Δrnh201Δ. Moreover, the addition of 20 mM hydroxyurea to growth media rescued the ts phenotype of dna2Δ405N, but failed to suppress the double mutants, dna2Δ405N rnh201Δ, dna2Δ405N rnh202Δ and dna2Δ405N rnh203Δ (Nguyen et al., 2011). Thus, the suppression of dna2 mutation also depends on a functional RNase H2, suggesting that RNase H2 plays a critical role in the removal of primer RNAs if cells have impaired Dna2. An alternative explanation, which is not mutually exclusive from the above possibility, is that the addition of 20 mM HU might have led to a decreased ratio of deoxyribonucleotides to ribonucleotides, causing a dramatic increase in ribonucleotide incorporation. This might render cells more dependent on the clean-up function of RNase H2 to remove misincorporated ribonucleotides present in newly synthesized DNA strands by replicative polymerases (Nick McElhinny et al., 2010a). The fact that Pol α misincorporates ribonucleotides more frequently than Pol δ or Pol ε is consistent with a more critical role of RNase H2 in lagging strand synthesis than in leading strand (Nick McElhinny et al., 2010b). It was shown that in humans, Rnh202-PCNA interaction is important to recruit RNase H2 to replication foci (Bubeck et al., 2011). Since the biochemical activity of RNase H2 is dedicated to the removal of ribonucleotide incorporated into DNA, the interaction between PCNA and RNase H2 may function to recruit RNase H2 to lagging strands for Okazaki fragment processing. It was also shown that elevated levels of misincorporated ribonucleotides during DNA replication cause genomic instability (Nick McElhinny et al., 2010a). Mutations in the human homologs of the three yeast RNase H2 subunits are related to the development of Aicardi-Goutieres syndrome (Crow et al., 2006).
5.2. Many stimulators of Dna2 and Fen1 to prevent formation of structural intermediates
MGS1 (Maintenance of Genome Stability 1) of S. cerevisiae was found to act as a multicopy suppressor of the ts growth defect of dna2Δ405N mutation (Kim et al., 2005). Mgs1 stimulated the structure-specific nuclease activity of yeast Fen1 in an ATP-dependent manner. ATP binding but not hydrolysis was sufficient for the stimulatory effect of Mgs1. Suppression of dna2Δ405N required the presence of a functional copy of RAD27. MGS1 is a highly conserved enzyme containing both DNA-dependent ATPase and DNA annealing activities, playing a role in post-replicational repair processes (Hishida et al., 2001 and 2002).
5.2.3. PCNA and RFC
RFC and PCNA are processivity factors for Pol δ and Pol ε. RFC, a clamp loader of PCNA, consists of five subunits (Rfc1 to 5) which share significant homology in seven regions referred to as RFC boxes (box II-VIII) (Cullman et al., 1995; Majka & Burgers, 2004). Although PCNA has been well known for its ability to stimulate Fen1 (Li et al., 1995; Tom et al., 2000; Frank et al., 2001; Gary et al., 1999; Gomes & Burgers, 2000), human RFC complex was recently found to markedly stimulate Fen1 activity via multiple stimulatory motifs per molecule (Cho et al., 2009). Fen1 stimulation by RFC is a separable function from ATP-dependent PCNA loading to primer ends. Analysis of stimulatory domain of RFC4 revealed that only a small part (RFC4170-194; subscripts indicate positions of amino acids) of it was sufficient to stimulate Fen1 activity and among them, the four amino acid residues were critical for Fen1 stimulation (Cho et al., 2009). The multiple stimulatory motifs present in the RFC complex could contribute to more rapid formation of ligatable nicks as an integral part of replication machinery while it moves along with replication forks (Masuda et al., 2007).
Mus81-Mms4 is a structure-specific endonuclease that can cleave nicked Holliday junctions, D-loop, replication forks, and 3’-flaps that could arise in vivo during the repair of damaged replication forks (Boddy et al., 2001; Kaliraman et al., 2001; Bastin-Shanower et al., 2003; Ciccia et al., 2003; Whitby et al., 2003). Overexpression of Mus81 suppressed the lethality of helicase-negative dna2K1080E (Kang et al., 2010) as well as dna2-2 and dna2-4, the two other dna2 mutant alleles isolated by others (Formosa & Nittis, 1999). In addition, Mus81-Mms4 and Fen1 stimulated each other in a manner requiring a specific protein-protein interaction. This indicates that the three endonucleases, Rad27, Mus81-Mms4, and Dna2, collaborate to remove a variety of structural intermediates in vivo.
5.2.5. Mph1 and Rad52
MPH1 was first identified as a mutator phenotype 1 gene (Entian et al., 1999), and the mph1Δ mutant displayed increased mutation rates and sensitivity to a variety of DNA damaging agents (Scheller et al., 2000). Based on this and other genetic studies, MPH1 is proposed to function in an error-free DNA damage bypass pathway that requires homologous recombination (Schürer et al., 2004). It was shown that Mph1 has DNA-dependent ATPase and 3’ to 5’ helicase activities (Prakash et al., 2005). Overexpression of Mph1 increased gross chromosomal rearrangements (GCR) by partially inhibiting homologous recombination through its interaction with RPA (Banerjee et al., 2008). These data suggest that Mph1 is important in maintaining the integrity of genome. MPH1 was isolated as a multicopy suppressor of dna2Δ405N and dna2K1080E. Purified Mph1 markedly stimulated the endonuclease activities of both Dna2 and Fen1 in vitro in an ATP-independent manner (Kang et al., 2009). Stimulation depends on the specific protein-protein interaction between the N-terminal domain of Dna2 and Mph1. Since overexpression of Mph1 also suppressed the dna2Δ405N mutant, the suppression of the Dna2 defect by Mph1 is due to the stimulation of Fen1 activity, and not of Dna2. Rad52 that mediates exchanging RPA with Rad51 in ssDNA is a multi-copy suppressor of dna2K1080E. Purified Rad52 is able to stimulate both Fen1 and Dna2 in vitro (Lee et al., 2011). The stimulation is independent of the recombination activity of Rad52.
5.3. Speculations on the presence of numerous stimulators of Dna2 and Fen1
In addition to the proteins mentioned above, the list of proteins that stimulate Fen1 and Dna2 is growing, which are most likely involved in maintenance of genome integrity. In humans, WRN, BLM, and RecQ5, the human homologues of yeast RecQ are an example of Fen1 stimulator (Brosh et al., 2001; Wang et al., 2005; Speina et al., 2010). Recently, it was shown that Dna2 and Pif1 can contribute to rapid nick formation by stimulating FEN1 (Henry et al., 2008). In addition, low levels of RPA also stimulated Fen1 activity particularly when short flaps were used as substrates. The acquisition of the ability of Fen1 or Dna2 to be stimulated by many proteins that work in close proximity may have conferred evolutionary benefits, because such an ability may permit faster generation and sealing of DNA nicks. Rapid generation and sealing of ligatable nicks may be more favorable in the preservation of genome integrity by converting unstable nicked DNA into stable duplex DNA.
5.4. Repair of faulty processing of Okazaki fragments
5.4.1. Homologous recombination as a last resort to repair faulty Okazaki fragments
When rad27-p (impaired interactions with PCNA) was combined with pol3-5DV (a mutant allele of a Pol δ subunit, defective in 3’ exonuclease and increased in displacement DNA synthesis), the double mutant cells were lethal in the absence of RAD51 that is essential for DSB repair (Jin et al., 2003). The lethal phenotype of rad27-p pol3-5DV rad51Δ was suppressed by overexpression of Dna2, suggesting that increased levels of long flaps resulting from mutant Pol δ required elevated levels of Dna2 for appropriate processing. In addition, the result above raises the possibility that excess levels of long flaps produced in rad27-p pol3-5DV cells could undergo DSB that can be harmlessly repaired by RAD51-dependent repair pathway. This idea is further supported by a number of genetic data. First, dna2-C2 mutant cells displayed extensive chromosomal fragmentation like cdc9 (DNA ligase 1) mutation in S. pombe (Kang et al., 2000). Second, rad27∆ rad52∆, dna2-1 rad27∆, dna2-1 rad52∆, dna2-2 rad52∆ double mutants are synthetic lethal (Jin et al., 2003; Budd et al., 2005). Third, ts dna2-22 mutant displayed increase in the rates of recombination and chromosome loss at non-permissive temperature (Fiorentino and Crabtree, 1997). Forth, the dna2-2 mutant cells showed hyperrecombination of rDNA, causing reduced life span of S. cerevisiae (Hoopes et al., 2002). In S. pombe, it was shown that functions of rhp51+ (recombination gene RAD51 homolog) were required for viability of dna2 mutants (Tsutsui et al., 2005). Moreover, Rad52 was isolated as a multi-copy suppressor of helicase-negative dna2K1080E. Rad52 plays a role in the formation of Rad51-ssDNA filament by exchanging RPA with Rad51 (Song and Sung, 2000). Thus, the mediator function of Rad52 is crucial to initiate strand invasion. The rad52-QDDD-308-311-AAAA (rad52-QDDD/AAAA) mutant cells failed to form MMS-induced DNA repair foci and were not able to repair MMS-induced damage (Plate et al., 2008). Moreover, the mutant Rad52-QDDD/AAAA protein barely interacted with RPA and showed inefficient recombination mediator activity while retaining wild type levels of DNA binding activity (Plate et al., 2008). The suppression of dna2 mutation by Rad52 required the mediator activity of Rad52; rad52QDDD/AAAA mutant was not able to suppress dna2K1080E (Lee et al., 2011). This suggests that faulty Okazaki fragment could lead to elevated levels of homologous recombination. In support of this, we discovered that dna2Δ405N showed increases in the rates of inter- and intra-chromosomal recombination and unequal sister chromatid recombination (Lee et al., 2011). Our results suggest that incomplete replication of lagging strand synthesis due to faulty processing of Okazaki fragments could be efficiently repaired via Rad52-dependent homologous recombination pathway (Fig. 4) (Reagan et al., 1995; Tishkoff et al., 1997b; Budd and Campbell, 2000). Recently, it was found that Dna2 itself is a critical player in DSB repair by directly participating in long-range resection of DSB ends in cooperation with Sgs1 in a redundant fashion with Exo1 (Mimitou and Symington, 2008; Zhu et al., 2008). Both helicase activity of Sgs1 and nuclease activity of Dna2 were essential for this resection, whereas the helicase activity of Dna2 was dispensable (Mimitou and Symington, 2008 and 2009; Zhu et al., 2008; Niu et al., 2010; Shim et al., 2010).
5.4.2. Roles of Mph1 and Mus81-Mms4 as structure managers
The involvement of Mph1, Mus81-Mms4, and Rad52 in Okazaki fragment processing is particularly interesting, not only because of their abilities to stimulate the endonuclease activity of Dna2 and/or Fen1, but also because of their roles in recombinational repair of lagging strand replication defect as suggested previously (Ii & Brill, 2005). We found that Mph1 is a multipurpose helicase that can unwind a variety of DNA structures such as junction structures containing three or four DNA strands. Mph1 is able to unwind fixed double-flap DNA (an intermediate form of equilibrating flaps) in such a way that among the two flaps the displacement of 5’ flap occurs first (Kang et al., 2011). Thus, the helicase activity of Mph1 could contribute to Okazaki fragment processing by facilitating conversion of equilibrating flaps into 5’ flaps, which are readily cleaved by Fen1. In addition, Mph1 was able to efficiently displace hairpin-containing oligonucleotides, as long as short (~5-nt) ssDNA regions were present at the ssDNA/dsDNA junction. The ability of Mph1 to displace 5’ secondary-structure flaps may allow cells to strip off the chronically problematic Okazaki fragments from the template, resulting in a gap equivalent in size to an Okazaki fragment, which can be filled in by Pol δ. Fen1 and Mus81-Mms4 appear to function in two separate processes because of their different substrate specificity (5’- and 3’-flap specific, respectively), the mutual stimulation observed in yeasts suggests a more direct inter-functional role between the two structure-specific endonucleases. The joint role of Fen1 and Mus81-Mms4 could come into effect via the interconversion between the substrates specific for each endonuclease. The 5’ or 3’ flap can be converted into a 3’ or 5’ flap, respectively, in a manner similar to that seen in Holliday junction migration. The equilibrating flaps (see Fig. 1. for structure) could be processed more rapidly if 5’ and 3’ flap specific enzymes could stimulate each other’s activity.
A helicase such as Mph1 could facilitate the interconversion process by virtue of its ability to displace the downstream strand. The product formed by this reaction would contain either 5’ or 3’ ssDNA flap depending on the polarity of the helicase involved, generating the structures suitable for cleavage by either Mus81-Mms4 or Fen1. Most likely candidates for such a function would include the helicases with branch migration activities such as WRN, RecQ1, and Mph1 (Prakash et al., 2009; Opresko et al., 2009; Burgreev et al., 2008). The human BLM helicase was shown to stimulate nuclease activity of the Mus81-Eme1 complex (Zhang et al., 2005). In addition, Rad54 was found to strongly stimulate Mus81-Mms4 in an ATP-independent manner in humans and yeasts (Matulova et al., 2009). Alternatively, a nuclease(s) that can simultaneously process both 5’ and 3’ double flaps could reduce the length of both flaps. This could more rapidly generate a DNA substrate that can be processed by either Fen1 or Mus81-Mms4. It was shown that endonuclease activity of human Dna2 is stimulated in the presence of double flaps (Kim et al., 2006).
6. Concluding remarks
Processing of Okazaki fragments is a complicated process at high risks of various types of DNA alterations such as base change, repeat expansion, and small duplications due to the involvement of anomalous structural DNA– a special type of DNA damages which, if left unrepaired, can promote genome instability. Examples of anomalous structure include nicks, unprocessed flaps, DSBs, and recombination intermediates. Formation of anomalous structures can be prevented by preemptive actions of Dna2 and/or by numerous ‘auxiliary’ factors that enhance endonuclease activities of Fen1 or Dna2. Alternatively, anomalous structures can be repaired by first forming DSBs, a key event that activates recombination. DSB-mediated recombination is regarded as the basis of genetic instability in eukaryotes since it can be a source of illegitimate recombination in higher organisms. A diverse array of auxiliary factors identified up to date may be a mirror image of a variety of structural intermediates present in vivo. The highly dynamic and capricious nature of structural intermediates renders correct processing of Okazaki fragments a formidable task which has to rely on a number of factors important for genome maintenance. Thus, Okazaki fragment processing is a platform where a number of proteins with roles in DNA replication and repair/recombination act together to minimize the hazardous outcome associated with its mechanisms in eukaryotes. In the future, the biggest challenge would be complete understanding of how each of the factors involved is regulated to fit into the complicated and dynamic network of protein-protein interactions required for failsafe processing of Okazaki fragments.
This work was supported by National Research Foundation of Korea (Grant No. 20100000009) funded by the Ministry of Education, Science and Technology.
Araki H. 2010 Cyclin-dependent kinase-dependent initiation of chromosomal DNA replication
Arudchandran A. Cerritelli S. M. Narimatsu S. K. Itaya M. Shin D. Y. Shimada Y. Crouch R. H. 2000 The absence of ribonuclease H1 or H2 alters the sensitivity of Saccharomyces cerevisiae to hydroxyurea, caffeine and ethyl methanesulphonate: implications for roles of RNases H in DNA replication and repair,Genes Cells 5 10 789 802
( Ayyagari R. Gomes X. V. Gordenin D. A. Burgers P. M. 2003). Okazaki fragment maturation in yeast. I. Distribution of fucntions between Fen1 and Dna2, J. Biol. Chem. 278 3 1618 1625.
Aviv T. Lin Z. Lau S. Rendl L. M. Sicheri F. CA Smibert 2003 The RNA-binding SAM domain of Smaug defines a new family of post-transcriptional regulators,
Bae S. H. Choi E. Lee K. H. Park J. S. Lee S. H. Seo Y. S. 1998 Dna2 of Saccharomyces cerevisiae possesses a single-stranded DNA-specific endonuclease activity that is able to act on double-stranded DNA in the presence of ATP,
Bae S. H. Seo Y. S. 2000 Characterization of the enzymatic properties of the yeast dna2 Helicase/endonuclease suggests a new model for Okazaki fragment processing,
Bae S. H. Bae K. H. Kim J. A. Seo Y. S. 2001a RPA governs endonuclease switching during processing of Okazaki fragments in eukaryotes,
Bae S. H. Kim J. A. Choi E. Lee K. H. Kang H. Y. Kim H. D. Kim J. H. Bae K. H. Cho Y. Park C. Seo Y. S. 2001b Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease,
Bae S. H. Kim D. W. Kim J. Kim J. H. Kim D. H. Kim H. D. Kang H. Y. Seo Y. S. 2002 Coupling of DNA helicase and endonuclease activities of yeast Dna2 facilitates Okazaki fragment processing,
Bae K. H. Kim H. S. Bae S. H. Kang H. Y. Brill S. Seo Y. S. 2003 Bimodal interaction between replication-protein A and Dna2 is critical for Dna2 function both in vivo and in vitro,
Banerjee S. Smith S. Oum J. H. Liaw H. J. Hwang J. Y. Sikdar N. Motegi A. Lee S. E. Myung K. 2008 Mph1p promotes gross chromosomal rearrangement through partial inhibition of homologous recombination
Bastin-Shanower S. A. Fricke W. M. Mullen J. R. Brill S. J. 2003 The mechanism of Mus81-Mms4 cleavage site selection distinguishes it from the homologous endonuclease Rad1-Rad10,
Bialek G. Grosse F. 1993 An error-correcting proofreading exonuclease-polymerase that copurifies with DNA-polymerase-alpha-primase,
Boddy M. N. Gaillard P. H. Mc Donald W. H. Shanahan P. Yates J. R. Russell P. 2001 Mus81-Eme1 are essential components of a Holliday junction resolvase,
Brosh R. M. Von Kobbe. C. Sommers J. A. Karmakar P. Opresko P. L. Piotrowski J. Dianova I. Dianov G. L. Bohr V. A. 2001Werner syndrome protein interacts with human flap endonuclease 1 and stimulates its cleavage activity,
Bubeck D. MA Reijns Graham S. C. Astell K. R. Jones E. Y. Jackson A. P. 2011 PCNA directs type 2 RNase H activity on DNA replication and repair substrates
ME Budd Campbell J. L. 1995 A yeast gene required for DNA replication encodes a protein with homology to DNA helicases,
ME Budd Choe W. C. Campbell J. L. 1995 DNA2 encodes a DNA helicase essential for replication of eukaryotic chromosomes
ME Budd Choe W. C. Campbell J. 2000 The nuclease activity of the yeast Dna2 protein, which is related to the RecB-like nucleases, is essential in vivo
ME Budd Tong A. Peng X. Polaczek A. Boone A. Campbell J. L. 2005 A network of multi-tasking proteins at the DNA replication fork preserves genome stability
Bugreev D. V. Brosh R. M. Mazin A. V. 2008 RECQ1 possesses DNA branch migration activity
Burgers P. M. Gerik K. J. 1998 Structure and processivity of two forms of Saccharomyces cerevisiae DNA polymerase delta,
Bullock P. A. Seo Y. S. Hurwitz J. 1991 Initiation of simian virus 40 DNA synthesis in vitro,
Cederberg H. Rannug U. 2006 Mechanisms of human minisatellite mutation in yeast
Ciccia A. Constantinou A. West S. C. 2003 Identification and characterization of the human Mus81-Eme1 endonuclease,
Cerritelli S. M. Crouch R. J. 2009 Ribonuclease H: the enzymes in eukaryotes
Cho I. T. Kim D. H. Kang Y. H. Lee C. H. Amangyelid T. Nguyen T. A. Hurwitz J. Seo Y. S. 2009 Human replication factor C stimulates flap endonuclease 1,
Conaway R. C. Lehman I. R. 1982 A DNA primase activity associated with DNA polymerase alpha from Drosophila melanogaster embryos,
Crow Y. J. Leitch A. Hayward B. E. Garner A. Parmar R. Griffith E. Ali M. Semple C. Aicardi J. Babul-Hirji R. Baumann C. Baxter P. Bertini E. Chandler K. E. Chitayat D. Cau D. Dery C. Fazzi E. Goizet C. MD King Klepper J. Lacombe D. Lanzi G. Lyall H. Martinez-Frias M. L. Mathieu M. Mc Keown C. Monier A. Oade Y. Quarrell O. W. Rittey C. D. Rogers R. C. Sanchis A. Stephenson J. B. Tacke U. Till M. Tolmie J. L. Tomlin P. Voit T. Weschke B. Woods C. G. Lebon P. Bonthron D. T. CP Ponting Jackson A. P. 2006Mutations in genes enconding ribonulease H2 subunits cause Aicardi-Goutieres syndrome and mimic congenital viral brain infection,
Cullmann G. Fien K. Kobayashi R. Stillman B. 1995 Characterization of the five replication factor C genes of Saccharomyces cerevisiae,
Dilcher M. Kohler B. von Mollard. G. F. 2001 Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6,
Entian K. D. Schuster T. Hegemann J. H. Becher D. Feldmann H. Güldener U. Götz R. Hansen M. CP Hollenberg Jansen G. Kramer W. Klein S. Kötter P. Kricke J. Launhardt H. Mannhaupt G. Maierl A. Meyer P. Mewes W. Munder T. Niedenthal R. K. Ramezani Rad. M. Röhmer A. Römer A. Hinnen A. et al. 1999 Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach,
Fiorentino D. F. Crabtree G. R. 1997 Characterization of Saccharomyces cerevisiae dna2 mutants suggests a role for the helicase late in S phase,
Formosa T. Nittis T. 1999 Dna2 mutants reveal interactions with Dna polymerase alpha and Ctf4, a Pol alpha accessory factor, and show that full Dna2 helicase activity is not essential for growth,
Frank P. Braunshofer-Reiter C. Wintersberger U. 1998Yeast RNase H(35) is the counterpart of the mammalian RNase HI, and is evolutionarily related to prokaryotic RNase HII,
Frank G. Qiu J. Zheng L. Shen B. 2001 Stimulation of eukaryotic flap endonuclease-1 activities by proliferating cell nuclear antigen (PCNA) is independent of its in vitro interaction via a consensus PCNA binding region,
Freudenreich C. H. Stavenhagen J. B. Zakian V. A. 1997 Stability of a CTG/CAG trinucleotide repeat in yeast is dependent on its orientation in the genome
Freudenreich C. H. Kantrow S. M. Zakian V. A. 1998 Expansion and length-dependent fragility of CTG repeats in yeast,
Garg P. Stith C. M. Sabouri N. Johansson E. Burgers P. M. 2004 Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication,
Garg P. Burgers P. M. 2005 How the cell deals with DNA nicks,
Gary R. MS Park Nolan J. P. Cornelius H. L. Kozyreva O. G. Tran H. T. Lobachev K. S. MA Resnick Gordenin D. A. 1999 A novel role in DNA metabolism for the binding of Fen1/Rad27 to PCNA and implications for genetic risk,
Gomes X. V. Burgers P. M. 2000 Two modes of FEN1 binding to PCNA regulated by DNA
Harrington J. J. Lieber M. R. 1994 The characterization of a mammalian DNA structure-specific endonuclease,
Henry R. A. Balakrishnan L. Ying-Lin S. T. Campbell J. L. Bambara R. A. 2010 Components of the secondary pathway stimulate the primary pathway of eukaryotic Okazaki fragment processing
Hishida T. Iwasaki H. Ohno T. Morishita T. Shinagawa H. 2001 A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability,
Hishida T. Ohno T. Iwasaki H. Shinagawa H. 2002Saccharomyces cerevisiae MGS1 is essential in strains deficient in the RAD6-dependent DNA damage tolerance pathway
Holmes J. Clark S. Modrich P. 1990 Strand-specific mismatch correction in nuclear extracts of human and Drosophila melanogaster cell linesProc. Natl. Acad. Sci. USA 87 15 5837 5841
Holmes A. M. Haber J. 1999 Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases,
Hoopes L. L. Budd M. Choe W. Weitao T. Campbell J. L. 2002 Mutations in DNA replication genes reduce yeast life span
Ishimi Y. Claude A. Bullock P. Hurwitz J. 1988 Complete enzymatic synthesis of DNA containing the SV40 origin of replication,
Jeong H. S. Backlund P. S. Chen H. C. AA Karavanov Crouch R. J. 2004 RNase H2 of Saccharomyces cerevisiae is a complex of three proteins
Jin Y. H. Obert R. Burgers P. M. Kunkel T. A. MA Resnick Gordenin D. A. 2001The 3’5’ exonuclease of DNA polymerase delta can substitute for the 5’ flap endonuclease Rad27/Fen1 in processing Okazaki fragments and preventing genome instability,
Jin Y. H. Ayyagari R. MA Resnick Gordenin D. A. Burgers P. M. 2003 Okazaki fragment maturation in yeast. II. Cooperation between the polymerase and 3’-5’-exonuclease activities of Pol delta in the creation of a ligatable nick,
Johansson E. Garg P. Burgers P. M. 2004 The Pol32 subunit of DNA polymerase delta contains separable domains for processive replication and proliferating cell nuclear antigen (PCNA) binding,
Johnson R. E. Kovvali G. K. Prakash L. Prakash S. 1995 Requirement of the yeast RTH1 5’ to 3’ exonuclease for the stability of simple repetitive DNA
Kaliraman V. Mullen J. R. Fricke W. M. Bastin-Shanower S. A. Brill S. J. 2001 Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease,Genes Dev. 15 20 2730 2740
Kang H. Y. Choi E. Bae S. H. Lee K. H. BS Gim Kim H. D. Park C. Mac Neill. S. A. Seo Y. S. 2000 Genetic analyses of Schizosaccharomyces pombe dna2(+) reveal that dna2 plays an essential role in Okazaki fragment metabolism,
MJ Kang Lee C. H. Kang Y. H. Cho I. T. Nguyen T. A. Seo Y. S. 2010 Genetic and functional interactions between Mus81-Mms4 and Rad27
Kang Y. H. MJ Kang Kim J. H. Lee C. H. Cho I. T. Hurwitz J. Seo Y. S. 2009 The MPH1 gene of Saccharomyces cerevisiae functions in Okazaki fragment processingJ. Biol. Chem. 284 16 10376 10386
Kang Y. H. Lee C. H. Seo Y. S. 2010 Dna2 on the road to Okazaki fragment processing and genome stability in eukaryotes
Kang Y. H. Munashingha P. R. Lee C. H. Nguyen T. A. Seo Y. S. 2011The DNA helicase activity of Mph1 has diverse roles in genome maintenance (in submission)
Kao H. I. Veeraraghavan J. Polaczek P. Campbell J. L. Bambara R. A. 2004 On the roles of Saccharomyces cerevisiae Dna2p and Flap endonuclease 1 in Okazaki fragment processing,
Kim J. H. Kang Y. H. Kang H. J. Kim D. H. Ryu G. H. MJ Kang Seo Y. S. 2005 In vivo and in vitro studies of Mgs1 suggest a link between genome instability and Okazaki fragment processing
Kim J. H. Kim H. D. Ryu G. H. Kim D. H. Hurwitz J. Seo Y. S. 2006 Isolation of human Dna2 endonuclease and characterization of its enzymatic properties
Kokoska R. J. Stefanovic L. Tran H. T. MA Resnick Gordenin D. A. Petes T. D. 1998 Destabilization of yeast micro- and minisatellite DNA sequences by mutations affecting a nuclease involved in Okazaki fragment processing (rad27) and DNA polymerase delta (pol3-t),
Kolodner R. D. Marsischky G. T. 1999 Eukaryotic DNA mismatch repair,
Kovtun I. V. Mc Murray C. T. 2008 Features of trinucleotide repeat instability in vivo
Kucherlapati M. Yang K. Kuraguchi M. Zhao J. Lia M. Heyer J. Kane M. F. Fan K. Russell R. Brown A. M. Kneitz B. Edelmann W. Kolodner R. D. Lipkin M. Kucherlapati R. 2001Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression, Proc. Natl. Acad. Sci. USA 99 15 9924 9929
Kunkel T. A. Erie D. A. 2005DNA mismatch repair,
Kunkel T. A. 2009Evolving views of DNA replication (in)fidelity,
Lee C. H. Shin Y. K. Phung T. T. Bae J. S. Kang Y. H. Nguyen T. A. Kim J. H. Kim D. H. MJ Kang Bae S. H. Seo Y. S. 2010Involvement of Vts1, a structure-specific RNA-binding protein, in Okazaki fragment processing in yeast, Nucleic Acids Res. 38 5 1583 1595
Lee C. H. Munasingha P. R. MJ Lee Huong P. T. T. Seo Y. S. 2011Recombination-mediated repair of faulty Okazaki fragment processing (manuscript in preparation)
Lee K. H. Kim D. W. Bae S. H. Kim J. A. Ryu G. H. Kwon Y. N. Kim K. A. Koo H. S. Seo Y. S. 2000The endonuclease activity of the yeast Dna2 enzyme is essential in vivo,
Ii M. Brill S. J. 2005Roles of SGS1, MUS81, and RAD51 in the repair of lagging-strand replication defects in Saccharomyces cerevisiae, Curr. Genet. 48 4 213 225
Li X. Li J. Harrington J. Lieber M. R. Burgers P. M. 1995Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen, J. Biol. Chem. 270 38 22109 12
Liu Y. Kao H. I. Bambara R. A. 2004Flap endonuclease 1: a central component of DNA metabolism,
Lopes J. Debrauwère H. Buard J. Alain Nicolas. A. 2002Genome Stability and DynamicsInstability of the human minisatellite CEB1 in rad27 and dna2-1 replication-deficient yeast cells,
Lopes J. Ribeyre C. Nicolas A. 2006Complex minisatellite rearrangements generated in the total or partial absence of Rad27/hFEN1 activity occur in a single generation and are Rad51 and Rad52 dependent,
Mac Neill. S. A. 2001DNA replication: partners in the Okazaki two-step,
Maga G. Villani G. Tillement V. Stucki M. Locatelli G. A. Frouin I. Spadari S. Hübscher U. 2001Okazaki fragment processing: modulation of the strand displacement activity of DNA polymerase delta by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1,
Majka J. Burgers P. M. 2004The PCNA-RFC families of DNA clamps and clamp loaders,
Maleki S. Cederberg H. Rannug U. 2002The human minisatellites MS1, MS32, MS205 and CEB1 integrated into the yeast genome exhibit different degrees of mitotic instability but are all stabilised by RAD27, Curr. Genet. 41 5 333 341
Mass G. Nethanel T. Kaufmann G. 1998The middle subunit of replication protein A contacts growing RNA-DNA primers in replicating simian virus 40 chromosomes,
Masuda Y. Suzuki M. Piao J. Gu Y. Tsurimoto T. Kamiya K. 2007Dynamics of human replication factors in the elongation phase of DNA replication, Nucleic Acids Res. 35 20 6904 6916
Masuda-Sasa T. Polaczek P. Peng X. P. Chen L. Campbell J. L. 2008Processing of G4 DNA by Dna2 helicase/nuclease and replication protein A (RPA) provides insights into the mechanism of Dna2/RPA substrate recognition,
Matulova P. Marini V. Burgess R. C. Sisakova A. Kwon Y. Rothstein R. Sung P. Krejci L. 2009Cooperativity of Mus81•Mms4 with Rad54 in the Resolution of Recombination and Replication Intermediates,
Mimitou E. P. Symington L. S. 2008Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing,
Mimitou E. P. Symington L. S. 2009Nucleases and helicases take center stage in homologous recombination,
Miret J. J. Pessoa-Brandão L. Lahue R. S. 1998Orientation-dependent and sequence-specific expansions of CTG/CAG trinucleotide repeats in Saccharomyces cerevisiae,
Modrich P. Lahue R. 1996Mismatch repair in replication fidelity, genetic recombination, and cancer biology,
Modrich P. 1997Strand-specific mismatch repair in mammalian cells,
Moe S. E. Sorbo J. G. Holen T. 2008Huntingtin triplet-repeat locus is stable under long-term Fen1 knockdown in human cells,
Murante R. S. Rust L. Bambara R. A. 1995Calf 5’ to 3’ exo/endonuclease must slide from a 5’ end of the substrate to perform structure-specific cleavage, J. Biol. Chem. 270 51 30377 30383
Nguyen T. A. Tak Y. S. Lee C. H. Kang Y. H. Cho I. T. Seo Y. S. 2011Functional reconstitution of Saccharomyces cerevisiae RNase H2 complex (manuscript in submission)
Nick Mc Elhinny. S. A. Kumar D. Clark A. B. Watt D. L. Watts B. E. Lundström E. B. Johansson E. Chabes A. Kunkel T. A. 2010aGenome instability due to ribonucleotide incorporation into DNA,
Nick Mc Elhinny. S. A. Watts B. E. Kumar D. Watt D. L. Lundström E. B. Burgers P. M. Johansson E. Chabes A. Kunkel T. A. 2010bAbundant ribonucleotide incorporation into DNA by yeast replicative polymerases,
Niu H. Chung W. H. Zhu Z. Kwon Y. Zhao W. Chi P. Prakash R. Seong C. Liu D. Lu L. Ira G. Sung P. 2010Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae,
Ohtani N. Haruki M. Morikawa M. Crouch R. J. Itaya M. Kanaya S. 1999Identification of the genes encoding Mn2+-dependent RNase I-III and Mg2+-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families, Biochemistry 38 2 605 618
Opresko P. L. Sowd G. Wang H. 2009The Werner syndrome helicase/exonuclease processes mobile D-loops through branch migration and degradation,
Parenteau J. Wellinger R. J. 1999Accumulation of single-stranded DNA and destabilization of telomeric repeats in yeast mutant strains carrying a deletion of RAD27,
Parenteau J. Wellinger R. J. 2002Differential processing of leading- and lagging-strand ends at Saccharomyces cerevisiae telomeres revealed by the absence of Rad27p nuclease,
Pavlov Y. I. Frahm C. Nick Mc Elhinny. S. A. Niimi A. Suzuki M. Kunkel T. A. 2006Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta,
CE Pearson Nichol Edamura. K. JD Cleary 2005Repeat instability: mechanisms of dynamic mutations,
Plate I. Hallwyl S. C. Shi I. Krejci L. Müller C. Albertsen L. Sung P. Mortensen U. H. 2008Interaction with RPA is necessary for Rad52 repair center formation and for its mediator activity,
Podust V. N. Hübscher U. 1993Lagging strand DNA synthesis by calf thymus DNA polymerases alpha, beta, delta and epsilon in the presence of auxiliary proteins,
Podust V. N. Podust L. M. Müller F. Hübscher U. 1995DNA polymerase delta holoenzyme: action on single-stranded DNA and on double-stranded DNA in the presence of replicative DNA helicases,
Prakash R. Krejci L. Van Komen S. Anke Schürer. K. Kramer W. Sung P. 2005Saccharomyces cerevisiae MPH1 gene, required for homologous recombination-mediated mutation avoidance, encodes a 3’ to 5’ DNA helicase
Prakash R. Satory D. Dray E. Papusha A. Scheller J. Kramer W. Krejci L. Klein H. Haber J. E. Sung P. Ira G. 2009Yeast Mph1 helicase dissociates Rad51-made D-loops: implication for crossover control in mitotic recombination,
Qiu J. Z. Qian Y. Frank P. Wintersberger U. Shen B. H. 1999Saccharomyces cerevisiae RNase H(35) functions in RNA primer removal during lagging-strand DNA synthesis, most efficiently in cooperation with Rad27 nuclease,
MS Reagan Pittenge C. Siede W. Friedberg E. C. 1995Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision-repair gene,
Remus D. Diffley J. F. X. 2009Eukaryotic DNA replication control: Lock and load, then fire,
Reynolds N. Warbrick E. Fantes P. A. Mac Neill. S. A. 2000Essential interaction between the fission yeast DNA polymerase delta subunit Cdc27 and Pcn1 (PCNA) mediated through a C-terminal 21Cip1)-like PCNA binding motif,
Rossi M. L. Pike J. E. Wang W. Burgers P. M. Campbell J. L. Bambara R. A. 2008Pif1 helicase directs eukaryotic Okazaki fragments toward the two-nuclease cleavage pathway for primer removal,
Rossi M. L. Bambara R. A. 2006Reconstituted Okazaki fragment processing indicates two pathways of primer removal,
Rossi M. L. Ghosh A. K. Bohr V. A. 2010Roles of Werner syndrome protein in protection of genome integrity,
Rydberg B. Game J. 2002Excision of misincorporated ribonucleotides in DNA by RNase H (type 2) and FEN-1 in cell-free extracts,
Ryu G. H. Tanaka H. Kim D. H. Kim J. H. Bae S. H. Kwon Y. N. Rhee J. S. Mac Neill. S. A. Seo Y. S. 2004Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast,
Scheller J. Schürer A. Rudolph C. Hettwer S. Kramer W. 2000MPH1, a yeast gene encoding a DEAH protein, plays a role in protection of the genome from spontaneous and chemically induced damage,
Schweitzer J. K. Livingston D. M. 1998Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturation, Hum. Mol. Genet. 7 1 69 74
Schürer K. A. Rudonph C. Ulrich H. D. Kramer W. 2004Yeast MPH1 gene functions in an error-free DNA damage bypass pathway that requires genes from Homologous recombination, but not from postreplicative repair,
Sclafani R. A. Holzen T. M. 2007Cell Cycle Regulation of DNA Replication,
Shen B. Singh P. Liu R. Qiu J. Zheng L. Finger L. D. Alas S. 2005Multiple but dissectible functions of FEN-1 nucleases in nucleic acid processing, genome stability and diseases,
Shim E. Y. Chung W. H. Nicolette M. L. Zhang Y. Davis M. Zhu Z. Paull T. T. Ira G. Lee S. E. 2010Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks,
Singh P. Zheng L. Chavez V. Qiu J. Shen B. 2007Concerted action of exonuclease and Gap-dependent endonuclease activities of FEN-1 contributes to the resolution of triplet repeat sequences (CTG)n- and (GAA)n-derived secondary structures formed during maturation of Okazaki fragments,
Song B. Sung P. 2000Functional interactions among yeast Rad51 recombinase, Rad52 mediator, and replication protein A in DNA strand exchange, J. Biol. Chem. 275 21 15895 15904
Speina E. Dawut L. Hedayati M. Wang Z. May A. Schwendener S. Janscak P. Croteau D. L. Bohr V. A. 2010Human RECQL5beta stimulates flap endonuclease 1,
Spiro C. Pelletier R. Rolfsmeier M. L. MJ Dixon Lahue R. S. Gupta G. MS Park Chen X. Mariappan S. V. Mc Murray C. T. 1999Inhibition of FEN-1 processing by DNA secondary structure at trinucleotide repeats,
Spiro C. Mc Murray C. T. 2003Nuclease-deficient FEN-1 blocks Rad51/BRCA1-mediated repair and causes trinucleotide repeat instability,
Tanaka H. Ryu G. H. Seo Y. S. Mac Neill. S. A. 2004Genetics of lagging strand DNA synthesis and maturation in fission yeast: suppression analysis links the Dna2-Cdc24 complex to DNA polymerase delta, Nucleic Acids Res. 32 21 6367 6377
Thomas D. C. JD Roberts Kunkel T. A. 1991Heteroduplex repair in extracts of human HeLa cells,
Tishkoff D. X. Filosi N. Gaida G. M. Kolodner R. D. 1997aA novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair,
Tishkoff D. X. Boerger A. L. Bertrand P. Filosi N. Gaida G. M. Kane M. F. Kolodner R. D. 1997bIdentification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2, Proc. Natl. Acad. Sci. USA 94 14 7487 7492
Tom S. Henricksen L. A. Bambara R. A. 2000Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1, J. Biol. Chem. 275 14 10498 10505
Tran P. T. Erdeniz N. Dudley S. Liskay R. M. 2001Characterization of nuclease-dependent functions of Exo1p in Saccharomyces cerevisiae,
Tsutsui Y. Morishita T. Natsume T. Yamashita K. Iwasaki H. Yamao F. Shinagawa H. 2005Genetic and physical interactions between Schizosaccharomyces pombe Mcl1 and Rad2, Dna2 and DNA polymerase alpha: evidence for a multifunctional role of Mcl1 in DNA replication and repair,
van den Broek. W. J. Nelen M. R. Van der Heijden G. W. Wansink D. G. Wieringa B. 2006Fen1 does not control somatic hypermutability of the (CTG)(n)*(CAG)(n) repeat in a knock-in mouse model for DM1,
Waga S. Stillman B. 1994Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro,
Wang W. Bambara R. A. 2005Human Bloom protein stimulates flap endonuclease 1 activity by resolving DNA secondary structure
Whitby M. C. Osman F. Dixon J. 2003Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4,
White P. J. Borts R. H. Hirst M. C. 1999Stability of the human fragile X (CGG)(n) triplet repeat array in Saccharomyces cerevisiae deficient in aspects of DNA metabolism, Mol. Cell. Biol. 19 8 5675 5684
Xie Y. Liu Y. Argueso J. L. Henricksen L. A. Kao H. I. Bambara R. A. Alani E. 2001Identification of rad27 mutations that confer differential defects in mutation avoidance, repeat tract instability, and flap cleavage,
Zhang R. Sengupta S. Yang Q. Linke S. P. Yanaihara N. Bradsher J. Blais V. Mc Gowan C. H. Harris C. C. 2005BLM helicase facilitates Mus81 endonuclease activity in human cells,
Zheng L. Jia J. Finger L. D. Gua Z. G. Zer C. Shen B. H. 2011Functional regulation of FEN1 nuclease and its link to cancer,
Zuo S. Bermudez V. Zhang G. Kelman Z. Hurwitz J. 2000Structure and activity associated with multiple forms of Schizosaccharomyces pombe DNA polymerase delta,
Zhu Z. Chung W. H. Shim E. Y. Lee S. E. Ira G. 2008Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends,