Maintenance of an accurate genome is critical for the survival of any cell. Cells face a large number of challenges to their genome. Some of these sources of damage include intrinsic such as the reactive oxygen species generated from oxidative respiration while others are exogenous such as the ultra violet rays from the sun 100. Irrespective of the source of genomic insults, cells have elaborate machineries devoted to repair specific kinds of DNA damage. Broadly speaking, one can classify DNA damage in following classes: i) base damage ii) nucleotide damage iii) replication induced DNA damage and iv) DNA breaks- single- and double-stranded 100. Depending upon the kinds of DNA damage, cells will recruit one (or many) of the following pathways to repair those damages. I will describe some of these pathways, briefly, in the following section:
i) Base damage- Base Excision repair, which involves removal of the mutated/altered base by a DNA glycosylase resulting in an abasic site. This abasic site is then cleaved by an AP endonuclease or an AP lyase followed by gap-filling by a DNA polymerase and ligation of the nick by a ligase 101.
ii) Nucleotide damage: Nucleotide Excision Repair (NER) is the pathway of choice for fixing nucleotide damage to DNA 101. NER can be broadly divided into global genomic NER (GG- NER) and transcription-coupled NER (TC-NER) 101. NER relies on identification of damaged nucleotides (primarily due to structural deviations in the DNA double helix) by specific protein(s) (Rad4-Rad23 in yeast and XPC in humans for GG-NER and elongating RNA Polymerase II for TC-NER) 101. Subsequently, the damaged region is accessed by general transcription factor, TFIIH, and XPG followed by XPA and XPF, resulting in the removal of about 21-23 nucleotides from the damaged region 101. DNA polymerases and ligases fill in the
damaged region 101. If the nucleotide damage occurs in a transcribed region, the rate of repair for this kind of damage is significantly faster and relies on the identification of the damage by elongating RNAPII 101. An additional factor (Rad26 in yeast and CSB in humans) gets recruited to the damaged area of the transcribed genome and results in coupling the TC-NER with GG- NER 101.
iii) Replication mismatches: DNA replication is intrinsically a high-fidelity process, but it can introduce nucleotide mismatches 102. The mismatched nucleotides, if not repaired during the process of replication itself, recruit the mismatch repair process. Unrepaired mismatches increase the mutation rates in the genome, resulting sometimes in the death of the cells 102. In addition, mismatches serve a useful purpose of increasing the potential for genetic variability for cells, by increased mutagenic potential. It is not surprising then that disruption of mismatch repair appears to be a favored mechanism for cancer evolution such as for colon cancer (about 50% hereditary non-polyposis colon cancer is due to mutations in MSH2 and MLH1 genes, that perform critical functions in mismatch repair) 102. Briefly, mismatch repair relies on multiple proteins that recognize the mismatch and excise the mismatched regions as well as components of replication machinery. The fundamental mechanism for mismatch repair is conserved from E.coli to humans, with minor details and regulatory features being different 102.
iv)Double strand break: Double strand breaks (DSBs) in DNA are one of the most severe forms of DNA damage. DSBs can occur in a programmed or an un-programmed fashion 103. An example of programmed DSB is the generation of antibody diversity in immune cells. Another example is during generation of male or female gametes through the process of meiosis 103. In addition, cells face a wide variety of insults that can cause DSBs such as ionizing radiation and DSBs induced by reactive oxygen species. Generally speaking two, mutually exclusive,
pathways repair DSBs 103: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ relies on the recruitment of yKu70-80 complex to the DSB, with the help of the Mre11-Rad50-Xrs2 (Nbs1) (MRX/MRN) complex 103. Recruitment of yKu70-80 results in the DSB ends being held together in physical proximity, in a relatively stable manner 103. The DSB ends can then be re-ligated after limited end processing (if necessary) by the help of additional NHEJ factors such as Lig4 and repair-associated DNA polymerases 103. Homologous recombination (HR), on the other hand, relies on the stable association of the MRX/MRN complex with the DSB ends 103. Such a stable association is followed by the recruitment of end- processing factors such as Sae2 (CtIP in mammals), which promotes 5’ à 3’ exonuclease activity 103. Such end-processing results in resected DNA ends, a particularly preferable substrate for the single strand DNA binding protein, RPA 103. Subsequently, the RPA is exchanged for Rad51, in terms of coating ssDNA. This Rad51-DNA complex is then used for homology search, followed by recruitment of a vast array of other proteins that help in using the homologous DNA strand as a template to repair the DSB 103. The decision to use one DSB repair pathway or another is one of the most crucial decisions that the cell makes in terms of repairing DNA damage 103. This decision is based on the cell cycle stage of the cell, and availability of regulatory factors 103,104. If a cell is in G1-phase, due to lack of CDK1 activity and absence of an additional homologous copy of DNA (for haploid genomes like budding yeast), cells use NHEJ to repair the DSB 103,104. If the cell is in S-G2/M stage, there is ample activity of cyclin- dependent kinases (primarily CDK1, which activates the exonuclease activity of Sae2/CtIP), which tilts the balance in favor of HR 103,104. In addition to the cell cycle stage, yKu70-80 acts as a repellent for recruitment of Sae2/CTIP to DSBs and hence inhibits HR 103,104. In mammalian cells, additional levels of regulation exist which depends upon 53BP1 and BRCA1 103,104.
BRCA1 is associated with breast cancer and 53BP1 was discovered as a p53 binding protein 105. It was subsequently discovered that the major function of 53BP1 is to regulate pathway choice after DSB in mammalian cells 105. 53BP1 gets recruited to the chromatin, depending upon the status of histone methylation (specifically H4K20me2) and occludes the access of BRCA1 to the DSB 105. BRCA1 associates with resection enzymes and thus 53BP1 inhibits the first step required for diverting the DBS through HR pathway 105. Recently additional factors have been discovered that contribute to the decision-making process after DSB. One of them is Rif1 (Rap1- interacting factor 1), which functions through binding to 53BP1 and inhibiting the recruitment of BRCA1 to the DSB, thus predisposing the DSB to be repaired by NHEJ 106-108. Inappropriate pathway choice can have disastrous consequences for the cell. For example, if a haploid cell decides to perform HR during G1 phase (like budding yeast, S.cerevisiae), it will likely end up using a region of the genome with poor homology and thus increase the genomic instability (due to strand exchange reactions that occur during HR) 103. To repair any kind of DNA damage, cells need to sense the DNA damage and then mount a response to repair it. This coordinated activation of signaling cascade is called DNA damage checkpoint, a focus of the following section.