BER components from sugarcane.
The base excision repair (BER) pathway has been associated with genome integrity maintenance. Owing to its central role, BER is present in all three domains of life. The studies in plants, considering BER, have been conducted using Arabidopsis and rice models. Therefore, future studies regarding BER are required in other organisms, particularly in crops such as sugarcane, to understand its mechanism, which may reflect the uniqueness of DNA repair in monocots. Our previous results have revealed that sugarcane is an interesting plant for studying this pathway considering the polyploidy genome and genome evolution. This chapter aimed to characterize the BER pathway in sugarcane by using different bioinformatics tools, for example, screening for BER homologs in the sugarcane genome to identify its members. Each sequence obtained was subjected to structural analysis, and certain differences were identified when Arabidopsis was compared to other monocots, including sugarcane. Moreover, ROS1, DEM, and DML3 were not identified as a complete sequence in the sugarcane EST database. Furthermore, FEN1 is present as two sequences, namely FEN1A and FEN1B, both featuring different amino acid sequence and motif presence. Furthermore, FEN1 sequence was selected for further characterization considering its evolutionary history, as sequence duplication was observed only in the Poaceae family. Considering the importance of this protein for BER pathway, this sequence was evaluated using protein models (3D), and a possible conservation was observed during protein–protein interaction. Thus, these results help us understand the roles of certain BER components in sugarcane, and may reveal the aspects and functions of this pathway beyond those already established in the literature.
- Saccharum spp.
- DNA repair
The base excision repair (BER) pathway is linked to the maintenance of genome integrity since BER is an essential genome defense pathway, which acts over a broad range of DNA lesions induced by endogenous or exogenous genotoxic agents . Owing to its central role, BER is present in all three domains of life . As a complex process, BER initiated by the excision of damaged base, proceeds through a sequence of reactions that generate various DNA intermediates and finish with the repair of the initial DNA structure. Nevertheless, BER focuses on repair, deals with DNA demethylation and erases the epigenetic mark 5-methycytosine (5mC) and converts it to cytosine . Thus, an emerging and crucial role of BER in epigenetic regulation is being investigated and characterized [4, 5, 6, 7]. Although various studies have been conducted in animal and microbial systems, BER knowledge regarding plants has been neglected.
Despite these apparent differences in plant research compared to other organisms, knowledge about the BER pathway in plants has gained immense interest in recent years. The results obtained so far reveal that plants possess orthologues of most BER genes previously found in other organisms [8, 9, 10]; however, they also retain some plant-specific BER proteins as well as distinct enzyme combinations not observed in other kingdoms (review by ). Unfortunately, most of these findings were based on the model
Furthermore, research has been conducted using the sugarcane expressed sequence tags (ESTs) project (SUCEST), which has identified possible DNA repair genes [17, 18]. BER sequences were predicted, although these investigations were conducted more than 10 years ago . Since then, there have been several improvements in bioinformatics tools as well as in sugarcane genome sequencing [20, 21, 22, 23, 24].
More studies are required to unravel the specific features of BER pathway in sugarcane, which may reflect the uniqueness of DNA repair in monocots. A new screening for BER homologs in the sugarcane genome was developed to gain advanced knowledge of BER in this crop. Each sequence was structurally analyzed. Thereafter, some of these sequences were selected for further investigating their evolutionary history. Tri-dimensional models have also been created to verify the conservation of mechanisms and protein–protein interactions in sugarcane BER components. The intriguing results displayed in this chapter raise questions regarding the roles of certain components of BER in sugarcane, just as in monocots, and they might broaden the aspects and functions of this pathway beyond those already established in the scientific literature.
2. Identification of base excision repair’s components in sugarcane
The BER components were identified in sugarcane through homology with the bioinformatic tools. In this regard, the SUCEST-FUN database, which assembles distinct sugarcane databases such as the Sugarcane Expressed Sequence Tags genome project (SUCEST-FUN) (http://sucest.lad.ic.unicamp.br/en/) ; Sugarcane Gene Index (SGI); SUCAST catalogs and SUCAMET, which include expression data (http://sucest-fun.org); GRASSIUS database  and records of the agronomic, physiological and biochemical characteristics of sugarcane cultivars, were used.
Sugarcane BER components identified were compared with sequences belonging to
|BER component||% Identity (Sb/At)||Protein name||Substrate or function||Role in BER||SUCEST-FUN ID|
|DEM, ROS1 and DMLE||(94.54/54.88, 51.79, 55.05)*||DEMETER, Repressor of silencing 1 and DEMETER-like protein 3||5-methylcytosine (5-meC)||Involved in the initial stage of BER, recognizing the damaged base.||comp89337_c0_seq1|
|OGG1||(96.64/59.29)||8-oxoguanine-DNA glycosylase 1||8-oxoguanine (8-oxoG)||comp78469_c0_seq3|
|NTH1||(97.85/ 54.09, 49.31)**||Endonuclease III homolog 1||oxidized pyrimidine||comp79344_c0_seq1|
|UDG||(96/57)||Uracil-DNA glycosylase||Uracil||comp64547_c0_seq4 and SCEQFL5048B07.g|
|MBD4L||(88.3/47)||Methyl-CpG-binding domain protein 4-like protein||G:T mismatches within methylated and unmethylated CpG sites.|
Uracil or 5-fluorouracil in G:U mismatches.
|MUTM (1 and 2)****||(94.49/68.64)|
|Formamidopyrimidine-DNA glycosylase||oxidation products of 8-oxoguanine (8-oxoG)||comp85541_c0_seq1;|
|ARP1 (1 and 3)****||(95.09/59.60)|
|DNA-(apurinic or apyrimidinic site) endonuclease||Ap site||Repair by-products (AP site) of BER or oxidation.||comp79331_c0_seq10;|
|FEN1 (A and B)****||(96/81.69) (82.97/73.07)||Flap endonuclease 1||5′ flap||Involved with the BER’s long-patch.||comp79282_c1_seq1 and SCEPRZ1008D03.g; comp85461_c0_seq2 and comp79282_c1_seq1|
|Pol λ||(95.16/54.70)||DNA POLIMERASE LAMBDA||Resynthesize missing nucleotides||It replaces the Polymerase beta acting on the BER short-patch.||comp80417_c0_seq9|
|TDP1||(95/45.7)||Tyrosyl-DNA phosphodiesterase 1||Processing of diverse 3′- and 5′-blocking groups at DNA ends||Processing of intermediate BER products||comp89039_c1_seq9 and comp89039_c1_seq3|
|LIG1||(91.67/70.96)||DNA ligase 1||Seal 5′-PO4 and 3′-OH polynucleotide ends||Involved in the long and BER’s short patch.||comp86584_c0_seq5 and comp86584_c0_seq7|
|LIG4||(97.53/73.25)||DNA ligase 4||Proposed to be involved in BER’s short-patch.||comp85403_c0_seq6|
|ZDP||(94.8/43.2)||Polynucleotide 3′-phosphatase ZDP||3′-phosphopolynucleotide||Processing of intermediate BER products.||comp78030_c0_seq1|
|PCNA||(100/85.55 and 86.69) ***||PROLIFERATING CELL NUCLEAR ANTIGEN||A scaffold to recruit the proteins involved in DNA replication, DNA repair, chromatin remodeling, and epigenetics||Involved in the BER’s long-patch.||comp82119_c0_seq2 and SCCCCL3140F04.g|
|PARP1||(96.7/60.6)||Poly [ADP-ribose] polymerase 1||Uses NAD+ as a substrate, synthesizes and transfers ADP-ribose onto aspartic and glutamic acid residues of acceptor proteins||Protects the BER substrate, present in the BER’s long-patch.||comp82301_c0_seq8 and SCAGLB1070H02.g|
|PARP2||(95.5/53.1)||Poly [ADP-ribose] polymerase 2||Not essential for DNA repair in the BER pathway.||comp85410_c0_seq3 and SCJFRT1012D11.g|
|XRCC1||(97.11/48.2)||X-RAY REPAIR CROSSCOMPLEMENTING PROTEIN 1||interacting with APE1 and stimulating its AP endonuclease activity, prepares the DNA substrate for the DNA polymerase activities.||Involved in the BER’s short-patch.||comp81667_c0_seq2, SCVPFL4C09E05.g and SCEZSD1082B05.g|
|WRN||(93.9/41.5)||WERNER SYNDROME ATP|
|Helicase enzyme||Interacts with several BER proteins: FEN1, PolB and PARP1||comp74108_c0_seq1, SCUTAM2089F01.g and SCSFLR2031F05.g|
3. BER components—missing and differences
Sugarcane exhibits almost all components of the BER pathway, even though ROS1, DEM and DML3 were not identified as complete sequences. These DNA glycosylases, which play pivotal roles in epigenetic processes , have been well characterized [28, 29, 30, 31] and were found in SUCEST-FUN as a single sequence without functional domains. Nevertheless, this result does not indicate a missing enzyme; epigenetic regulation is crucial, particularly in plants, and even more in polyploids organisms [32, 33]. Furthermore, the sugarcane database compiles numerous fragmented sequences that were not assembled and functionally annotated yet, as most data were from the transcriptome .
In contrast, differences were observed between the sequences of grasses analyzed (sugarcane and
|Query||Accession||Protein domain||lenght (aa)|
|Interaction with PCNA|
|Interaction with PCNA|
|Interaction with PCNA|
|DNLI4_ARATH (Q9LL84)||cl36689||dnl1 superfamily||1219|
DNA ligase IV revealed certain differences regarding the domain disposition on the sequence (Table 2). Additionally, the sequences reveal variable identity (Table 1), thereby indicating high similarity within the grass plants. Notably, the BRCT domain is present in the sequences of
4. BER’s first step - base lesion recognition
BER is initiated by lesion-specific DNA glycosylases. The basic DNA glycosylase enzymatic process involves excision of the modified nucleobase from the DNA by catalyzing the hydrolysis of the N-glycosidic bond . Regarding sugarcane, some of the BER’s glycosylases were identified and characterized, suggesting the maintenance of the enzymes in
The DNA glycosylase OGG1 was identified in sugarcane and is called OGG1_CANA. This glycosylase as well as other sequences belonging to the
Sugarcane NTH1, called NTH1_CANA, belongs to the Helix-hairpin-Helix (HHH) superfamily . Furthermore, regarding
Another glycosylase identified was UDG_CANA, which was conserved in the domain belonging to the UDG superfamily, more precisely concerning family-1 [51, 52, 53]. Additionally, it conserved aspartic acid (D) as an active site . It is known that the human UNG gene encodes two forms of the protein, one directed towards the mitochondria (UNG1) and another towards the nucleus (UNG2) . The
Ramiro-Merina et al.  demonstrated that
5. AP site removal—AP endonuclease role in sugarcane
AP endonuclease is an essential enzyme for BER pathway as this enzyme identifies and process AP (apurinic/apyrimidinic) site . These AP sites may be a result of the action of DNA glycosylases or it may be spontaneously generated. Unrepaired AP sites can lead to mutations during semiconservative replication, which indicates the importance of the role of AP endonuclease in maintenance of the genetic code .
ScARP1 has greater similarity with AtARP (60%), whereas AtAPE1L and AtAPE2 reveal a correspondence below 50%. These values may indicate diversity in structure, amino acid composition, and perhaps function. ScARP3 reveals a divergence compared with ScARP1. ScARP3 is closer to AtARP, presenting an even higher percentage of identity (75%). Maíra et al.  demonstrated that the sequence ScARP3 would be closer to the group of dicotyledonous plants, whereas ScARP1 would be included within the monocots, more precisely together with representatives of the
Medeiros et al.  purified ScARP1 and verified the enzymatic activity of this sugarcane enzyme against several substrates. This study found the capability of ScARP1 to process AP sites; however, other enzymatic activities (exonuclease, phosphatase, and 3′-phosphodiesterase) were not confirmed. The AP endonuclease activity complementation assay in extracts of
6. Flap endonuclease (FEN1)—BER’s long-patch in sugarcane
FEN1 is a structure-specific nuclease that can remove flap structures and is involved in different DNA metabolic pathways, including DNA replication, DNA repair, apoptotic DNA degradation, and maintenance of telomere stability . In case of BER, FEN1 in complex with proliferating cell nuclear antigen (PCNA) plays a pivotal role in the long patch as it removes a short flap structure generated by Pol β activity .
Regarding plants, it is known that two FEN1 counterparts were identified in rice (
As observed in
Considering the protein structure, FEN1 is a nuclease that features two regions: the N-terminal region and I-region . The alignment of FEN1 from
7. PCNA role in plants
Studies on PCNA have revealed that it plays a crucial role in DNA replication as well as in DNA repair, cell cycle regulation and apoptosis [70, 71, 72]. In
Considering the difference between AtPCNAs, Anderson et al.  demonstrated that co-expression of POLH (DNA polymerase eta - Pol η) and AtPCNA2 (and not AtPCNA1) was necessary to restore normal resistance to UV radiation in the yeast RAD30 mutant. The difference was in lysine (K) 201 present in AtPCNA1, which would inhibit the ubiquination of lysine 164, thus affecting its connection with Pol η and not being able to act on trans-lesion synthesis (TLS) and restore the progression of the replication fork. The lysine at position 201 of AtPCNA1 belonged to the group comprising amino acids with electrically charged side chains. In the case of K, this could be endowed with a positive charge, whereas the corresponding one at AtPCNA2 would be an asparagine (N) that belonged to the group of amino acids with polar side chains without being loaded. In PCNA_CANA, the corresponding residue in question would be a glutamine that concerns the same group as N, which leads to the conclusion that sugarcane PCNA would be closer to AtPCNA2 than AtPCNA1 and could, as such, act in the TLS.
The three-dimensional model of sugarcane’s PCNA is revealed as homotrimeric architecture in the form of a ring, comprising three identical chains of PCNA, as indicated by different colors in the sugarcane PCNA model (Figure 2). Additionally, this model exhibits sequence and structural similarity with other PCNAs, as observed in Figure 2. Compared to
PCNA is generally called a sliding clamp, since it was predicted that the double strand of DNA would pass through the opening of the PCNA ring and would serve as an anchoring platform for several proteins involved in DNA metabolism . The attainable preservation of this function was verified in the
8. Sugarcane protein models—conservation throughout plants
PCNA and FEN1A were proteins identified in sugarcane, which were presumed to interact with each other . This is due to PCNA interaction sequence detected in the N-terminal segment of FEN1A. To verify the truthfulness of this interaction, three-dimensional models were created for PCNA and FEN1A sugarcane proteins. These models were assessed for the conservation of secondary structure, active sites, and residue interactions with the substrate. Based on this analysis, the role of these sugarcane proteins can be established.
PCNA, as previously mentioned, would serve as a scaffold, and moreover, various functions can be performed ranging from DNA methylation to base excision repair. Thus, using the IUL1 crystal that comprises the human PCNA associated with FEN1 , the possibility of the sugarcane’s predicted models of these proteins that may interact with each other was verified. This result demonstrates that FEN1 of sugarcane is associated with the homotrimeric ring of PCNA (Figure 4). The sequence of interaction with PCNA differs, revealing that this sequence is in the interface of PCNA and FEN1 interaction.
9. BER pathway—evolutionary analysis in the grass outlook
Overall, the phylogenetic analyses revealed differences in the presence or absence of duplication of BER pathway components. In few cases, duplication was observed in dicotyledons and not in monocotyledons, for example, NTH, PCNA and DNA ligase 1. Herein, structural difference was noted (size, presence or absence of certain conserved domains), indicating diverse DNA repair mechanisms between plants.
Singh et al.  compared the plant genomes available at that time, thus aiming to compare the genes involved in DNA repair and recombination. They found that FEN1, in the genome of monocotyledons (corn, rice,
A new analysis regarding FEN1 in plants, particularly sugarcane, was conducted. It was discovered that FEN1B was only found within
Although the absence of region may compromise the enzymatic activity of FEN1B, the other residues, domains and active sites were conserved. These findings raise questions regarding the maintenance of FEN1B in the genome of these organisms, its functions and its role in BER.
Maíra et al.  proposed that a whole genome duplication event (WGD) would be related to the duplication observed in the AP endonuclease sequence in the grasses group; however, further studies indicate that duplications are present in other plant groups in addition to
Notably, the fate of the vast majority of duplicate genes resulting from segmental duplication includes the nonfunctionalization of a member of the pair [80, 81],which should occur within a few million years in the absence of any intrinsic advantage of duplicate copying [81, 82]. Specifically, plant genomes, on average, reveal 65% of their annotated genes that are duplicated . Most of these copies are derived from ancient WGD events in the terrestrial plant lineage . Li et al.  investigated the fate of duplicate genes from 40 different species of flowering plants; of these, all species experienced at least one or more WGD events throughout their evolutionary history. The loss of genes was observed immediately after genome duplication, so that the genes quickly returned to the state of a single copy ; however, some of these genes have preserved their state of multiple copies. Such genes belong to families of genes involved in the response to biotic and abiotic stress, and are therefore important for the adaptation of the plant to the environment. Thus, it is possible to correlate the duplication and retention of these copies with an adaptive advantage such that genes can confer to the plant, allowing it to act more efficiently in response to environmental variations. DNA repair genes are linked to this hypothesis, since they are necessary to maintain the stability of the genome and preserve genetic information. In addition to the fact that several of these genes, already described in this chapter, act in other processes of adaptive importance such as response to oxidative stress.
In sugarcane as well as in other plants, except for the plant models, few studies have focused on the characterization and structural analysis of individual components of metabolic pathways. Moreover, it should be considered that the traditional breeding strategy lags behind the demand for commercial needs due to insufficient knowledge on characteristics related to stress tolerance, inefficient selection techniques and low genetic variation and fertility. The evident deficiency of biotechnology will be supplemented with studies aimed at the biochemical and functional characterization of important pathways and their components, such as the DNA repair pathway, for instance, the BER pathway. Therefore, it is necessary to emphasize the importance of this chapter in other plant species apart from sugarcane, provide supplementary information, and raise questions on the components of the BER pathway and its evolutionary issue regarding monocots and dicots.
The authors wish to thank to Coordenação de Aperfeiçoamento Pessoal de Nível Superior-CAPES, Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, and Ministério de Ciência, Tecnologia, Inovação (MCTI) for financial support.
Conflict of interest
The authors declare no conflict of interest.