Week 3_Lecture 1.pdf

Course Title: Genome Editing and Engineering Module 3 : Recombination Lecture 1: Homologous and non- homologous recombination Prof. Utpal Bora...

Course Title: Genome Editing and Engineering Module 3 : Recombination Lecture 1: Homologous and non- homologous recombination Prof. Utpal Bora Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati Kamrup, Assam- 781039, India Email: [email protected] From our previous discussion you know that organisms are continuously exposed to a myriad of DNA damaging agents during its lifetime. You also now know that DNA damage are broadly of two types: (1) Endogenous damage caused by reactive oxygen species (ROS) that are derived from metabolic byproducts, replication errors, DNA base mismatches and topoisomerase-DNA complexes (2) Exogenous damage caused by radiation (UV, X-ray, gamma), hydrolysis, plant toxins, and viruses. If left unrepaired DNA damage impact health and modulate disease-states. Luckily, robust DNA repair and damage-bypass mechanisms sincerely protect the DNA by either removing or tolerating the damage to ensure an overall survival. Genetic or metabolic diseases occur when the repair mechanism fails for any reason which is an exception rather than a usual event. Organisms respond to DNA damage at the cellular and molecular level by instigating robust DNA damage response (DDR) pathways, that physically remove or repair the damage in a substrate- dependent manner. There are at least five major known DNA repair pathways active throughout different stages of the cell cycle, allowing the cells to cope up with the inflicted the DNA damage. base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), homologous recombination (HR) and non-homologous end joining (NHEJ) A few other specific lesions can also be removed by direct chemical reversal and interstrand crosslink (ICL) repair. DSBs are considered as the most toxic lesion. Double-strand DNA breaks (DSBs) are generated by various ways, i. Endogenous stresses resulting from cellular metabolism, such as replication stress and reactive oxygen species (ROS), ii. Exogenous factors, such as ionizing radiation and chemotherapy agents (e.g., topoisomerase inhibitors). iii. DSBs may also arise through errors in DNA replication or as normal intermediates during programmed cellular processes such as meiosis or V(D)J recombination (which generates mature immunoglobulin or T-cell receptor genes from the separate fragments of the germline genome). DSBs can also be programmed to trigger beneficial genomic rearrangements during meiotic differentiation or the establishment of the immune system. Adapted from https://encyclopedia.pub/entry/9091 Strategies for repairing double-strand breaks. Whenever DSBs occur and by whatever process, these breaks must be repaired to ensure survival of the cell. i. Homologous recombination. In this strategy, a double-strand break present in one chromatid is repaired using its intact sister chromatid as a template. The repair of breaks by homologous recombination is a high fidelity process as it ensures that all the genetic information at the break site is retained. ii. Non-homologous end joining. NHEJ involves the simple rejoining of the broken DNA ends, regardless of the DNA sequence. This mechanism is error-prone, as small deletions may be introduced at the break site. iii. Alternative form of NHEJ and is frequently abbreviated as A-NHEJ, or simply A-EJ Front. Cell Dev. Biol., 12 July 2021 | https://doi.org/10.3389/fcell.2021.708763 Copyright © 2021 Ackerson, Romney, Schuck and Stewart. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).  Key decision points in the repair of DSBs (1) The DNA ends are bound by either MRN or the Ku70/80 heterodimer. Binding and retention of MRN will shift repair toward HR and the binding of Ku shifts repair toward to NHEJ. (2) Short range resection by MRN. (3) Long-range resection of the DNA and RPA binding. (4) RPA is exchanged for RAD51, which facilitates strand invasion, DNA synthesis and HR repair. Front. Cell Dev. Biol., 12 July 2021 | https://doi.org/10.3389/fcell.2021.708763 Copyright © 2021 Ackerson, Romney, Schuck and Stewart. This is an open-access article distributed under the terms of the Creative Commons Attribution License. Homologous recombination promotes the pairing between identical or nearly identical DNA sequences and the subsequent exchange of genetic material between them. Homologous recombination is termed as the guardian of genome integrity, as it acts to repair DNA damage. Homologous recombination is found to be are involved in rescue replication forks that have stalled for various reasons, such as a missing factor (e.g., the helicase), or a particular difficulty upstream of the fork, such as supercoiling or intense traffic of proteins. Homologous recombination is a highly conserved process from bacteria to humans, that serves to repair double-stranded breaks or single-stranded gaps in the DNA. For eg. in higher organisms, the Rad51 protein is a structural and functional homologue of the bacterial strand-exchange protein RecA. HR is also a driving force for the evolution of multigene families. Homologous recombination has played a major role in evolution and genome dynamics, by changing gene copy numbers through deletions, duplications, and amplification. Intrachromosomal recombination between ribosomal operons or between mobile elements scattered into the genome leads to deletion or tandem duplications of large regions within the genome, up to several hundred kilobases. The emerging technical application of HR constitutes the basis of targeted gene replacement for gene therapy as well as for the precise design of engineered organisms. The basic model for homologous recombination was largely derived from genetic studies in fungi such as Ustilago maydis and Saccharomyces cerevisiae Studies with the bacterium Escherichia coli, have provided us crucial biochemical insights into the mechanism of homologous recombination. In E. Coli , about 20 genes are found to be involved in recombination. They produce, specific proteins which carry out each of the key steps in homologous recombination.  Let us examine the events following a DSB for Resection. Most molecular models of homologous recombination describe the process in three key steps: i. Strand exchange, ii. Branch migration and iii. Resolution. Current Biology 11, Issue 7, 3 April 2001, Pages R278-R280. https://doi.org/10.1016/S0960-9822(01)00138-5 Nature Reviews Molecular Cell Biology 7: 739-750. DOI:10.1038/nrm2008. This file is licensed under the Creative Commons Attribution-Share Alike 3.0 Unported license. Strand exchange, involves pairing of the broken DNA end with the homologous region of its sister chromatid, followed by strand invasion to form a DNA crossover or Holliday junction. This process generates regions of heteroduplex DNA comprising DNA strands from different sister chromatids. Resection: First there is 5′-to-3′ resection of the double-strand break (DSB), producing DNA ends with 3′ single-stranded DNA tails. Strand Invasion: The free 3′ ends invade a homologous DNA duplex, forming a DNA crossover or Holliday junction and act as a primer to initiate new DNA synthesis. Branch migration: Branch migration of the Holliday junction extends the region of heteroduplex away from the initial site of crossover. Branch migration. during branch migration, the Holliday junction is translocated along DNA, extending the region of heteroduplex away from the initial crossover site. In the last step the Holliday junction intermediate is resolved by cleavage of the junction to form separate duplex DNA molecules again Holliday Junction Resolution: Holliday junctions are resolved by endonucleolytic cleavage of either the crossed strands or non-crossed strands of the junction. Molecular Mechanisms of Repair DNA ends resulting from a double-strand break is processed by a multi-functional enzyme complex called RecBCD RecBCD is a sequence-regulated bipolar helicase- nuclease that splits the duplex into its component strands and digests them until it encounters Chi site which is a recombinational hotspot. The nuclease activity is then attenuated and RecBCD loads RecA onto the 3′ tail of the DNA. Nature 432: 187-193. doi: 10.1038/nature02988 (CC BY 3.0) A Chi (χ) site is a short stretch of DNA in the genome of a bacterium near which homologous recombination is more likely to occur than on average across the genome. For this reason Chi sites are also referred to as "recombination hot spots". Chi sites serve as stimulators of DNA double-strand break repair in bacteria and its sequence is unique to each group of closely related organisms; In enteric bacteria E. coli and Salmonella, the core sequence is 5'-GCTGGTGG-3’ In addition it includes about 4 to 7 nucleotides to the 3' side of the core sequence which are important. Primary structure of the RecBCD enzyme. The total number of amino acids (aa) in each polypeptide is indicated in parentheses. The RecB protein is modular. The N-terminal domain contains seven motifs characteristic of SF1 helicases. The C-terminal domain contains motifs characteristic of a diverse family of nucleases. The nuclease motif contains key catalytic aspartate and lysine residues. Microbiol Mol Biol Rev. 2008 Dec; 72(4): 642–671.  Primary structure of the RecBCD enzyme. Let us study each component one at a time The RecB protein contains 1180 aa residues and is modular. Nuc N C SF1 Helicase Nuclease The N-terminal domain contains The C-terminal domain contains nucleases Helicase function and has seven motifs. “Nuc” marks the position of characteristic SF1 motifs 1, 1a, 2, 3, nuclease motif 3, which contains key 4, 5, and 6. catalytic aspartate and lysine residues. Microbiol Mol Biol Rev. 2008 Dec; 72(4): 642–671.  Chi site N C N C SF1 Helicase The RecC protein has 1122 aa residues and The RecD protein with 608 aa residues contains the Chi recognition site. contains seven conserved SF1 helicase motifs (1, 1a, 2, 3, 4, 5 and 6). Microbiol Mol Biol Rev. 2008 Dec; 72(4): 642–671.  RecBCD Nuc N C SF1 Helicase Nuclease SF1 Helicase Rec B Chi site Rec D N C B Nuclease Chi site And RecA N C loading SF1 Helicase 1. RecC recognizes Chi sequence 2. Rec C signals Rec D to STOP Adapted from Genetics. 2016 Sep; 204(1): 139–152, 3. RecD stops and then signals RecB to cleave DNA Microbiol Mol Biol Rev. 2008 Dec; 72(4): 642–671. 4. RecB cleaves and continues unwinding DNA and loads RecA RecBCD-catalyzed DNA end-processing reaction. (1) RecBCD binds tightly to a blunt (or nearly blunt) DNA end of a linear DNA duplex. (2) RecBCD couples the hydrolysis of ATP to DNA translocation and unwinding (helicase activity). The ssDNA products are cleaved asymmetrically, with the degradation of the 3′-terminated ssDNA tail being much more vigorous than the degradation of the complementary tail. (3) The enzyme continues to translocate until it pauses at a correctly oriented Chi sequence. At the Chi sequence the biochemical properties of the enzyme are altered dramatically. After Chi recognition, RecBCD facilitates the loading of the RecA protein onto the 3′ ssDNA tail. Microbiol Mol Biol Rev. 2008; 72(4): 642–671. Nature 432: 187-193. doi: 10.1038/nature02988 doi: 10.1128/MMBR.00020-08 CC BY 3.0 (4) The enzyme continues to translocate, but the nuclease polarity is switched; the degradation of the 3′ ssDNA tail is attenuated, whereas the hydrolysis of the 5′ ssDNA tail is upregulated. (5) RecBCD repeatedly deposits RecA protomers, which act as nucleation points for filament growth primarily in the 5′→3′ direction. (6) The RecBCD enzyme dissociates from the DNA. The product of the enzyme is a recombinogenic nucleoprotein complex of the RecA protein bound to the 3′ ssDNA tail with Chi at its terminus. This product invade homologous duplex DNA to promote the recombinational repair of a DSB or to restart DNA replication. Nature 432: 187-193. doi: 10.1038/nature02988 CC BY 3.0 It has been found that the permanent inactivation of RecBCD enzyme by Chi sites in duplex DNA occurs by the disassembly of the enzyme into its three constituent subunits. It is hypothesized that this inactivation occurs in two distinct steps. 1. Upon encountering a Chi sequence, RecBCD enzyme undergoes its first change: it retains its ability to travel along the DNA and to cut a hairpin DNA structure at the distal end of the DNA but loses its ability to nick at subsequently encountered Chi sites on the same DNA molecule (Taylor and Smith 1992). 2. The second change, the disassembly of the enzyme into three inactive subunits, may occur either during continued unwinding beyond Chi or upon reaching the end of the DNA. Andrew F. Taylor and Gerald R. Smith. Genes & Dev. 1999. 13: 890-900 Protein: Protein RecA RecA is a protein of about 350 amino-acid residues. Its sequence is highly Gene: recA conserved among eubacterial species. It is also found in the chloroplast Organism: Escherichia coli (strain K12) of plants. Length: 353 RecA-like proteins are found in archaea and diverse eukaryotic Mass (Da): 37,973 organisms, like fission yeast, mouse or human. RecA is involved in homologous recombination and bypass mutagenic DNA lesions by the SOS response. It catalyzes the, i. ATP-driven homologous pairing and strand exchange of DNA molecules necessary for DNA recombinational repair ii. hydrolysis of ATP in the presence of single-stranded DNA, iii. ATP-dependent uptake of single-stranded DNA by duplex DNA, and iv. ATP-dependent hybridization of homologous single-stranded DNAs. (PubMed:22412352). https://www.uniprot.org/uniprot/P0A7G6  KRXKR ATP Binding site Hydrolysis NTD 1-33 66-73 ATP-ase domain 34-269 CTD 270-352 Motif L L 1 2 Site I dsDNA gateway Flexible Region D144 Site II Tail 329-352 157-216 270-328 24-37 Hydrolytic Residue Mg2+ 226-245 E96 binding Flexible region Mg2+ Binding L1 loop 157-164 L2 loop 195-209 Flexible region 272- 352 RecA is composed of 352 amino acids and contains three major structural domains: i. a central, core ATPase domain (CAD), which extends from the 34th to the 269th amino acid (in green), and two smaller ii. NTD and CTD, which extend from the 1st to 33rd (in red) and from the 270th to 352nd amino acids, respectively (in yellow). The ATPase core domain includes different sites: the ATP binding site (residues 66–73); site I, which is the ssDNA binding site (residues 157–216); and site II, which is the dsDNA binding site, (residues 226–245). It also includes residues responsible for the ATP hydrolysis activity (E96 and the [KR] × [KR] motif in positions 248–250). The CTD can be divided into two domains: the dsDNA gateway (residues 270– 328) and the CTD tail (residues 329–325) which modulates the protein activity. The Mg2+ binding is coordinated by D144 and CTD tail.  Flexible region 272- The 4 flexible regions of the protein are depicted by violet boxes. 352 These regions are the regions between residues 24 and 37, at the end of the N-terminal domain (NTD) and the beginning of the core domain), 157 and 164 (loop L1), 195 and 209 (loop L2) and 270 and 352 (CTD). RecA activity ssDNA and RecA along with its cofactor ATP form an active right-handed helical nucleoprotein filament with six RecA monomers per turn. This active nucleoprotein filament searches for and captures a homologous dsDNA to produce a synaptic structure. Once a region of homology is found, the ssDNA strands on the homologous chromosomes are exchanged, producing heteroduplex DNA. The nucleoprotein filament has hydrolytic activity, and this hydrolysis is carried out by the [KR] × [KR] hydrolysis motif containing Lys248 and Lys250, which co-operate with Glu96 on the other monomer. 1. The single-stranded DNA (ssDNA) binds to site I of the protein, forming a nucleoprotein filament. 2. Then, ATP, a RecA cofactor, binds to the ATP binding site and activates the filament. 3. After that, RecA performs a homology search to find a homologous double-strand (dsDNA). In this process, dsDNA interacts first with the NTD and then 4. with the CTD, by which it can move to site II, 5. where it binds dsDNA. 6. If the bound dsDNA is homologous to the ssDNA, strand exchange is performed; if it is not, the dsDNA is released. Homologous Recombination in eukaryotes 2.1. Resection Sensor of DSBs: Double-strand breaks are recognized by the Mre11– Rad50–Nbs1 (MRN) or Mre11 complex. Capture of DNA ends by the MRN complex leads to the rapid activation of the ataxia-telangiectasia mutated (ATM) kinase which phosphorylates diverse substrates participating in DNA damage response. BRCA1 and p53-binding protein 1 (53BP1)- has opposing roles and influences the choice of homologous recombination over non-homologous Cancers 2021, 13(9), 2083; https://doi.org/10.3390/cancers13092083 end joining and potentially other mutagenic This is an open access article distributed under the Creative Commons Attribution License pathways of DSB repair. Apart from i) Initiation of resection BRCA1 also helps in ii) Loading of RAD51. 2.2. Loading RAD51 on ssDNA, Search for Homology and Strand Invasion The 3′ ssDNA stretch created by resection is coated with replication protein A (RPA), protecting it. The loading of RAD51 onto ssDNA is performed by the BRCA2-PALB2 complex. This protein complex interacts with BRCA1 and catalyzes the replacement of RPA by RAD51 on the stretch of 3′ ssDNA, creating the RAD51-ssDNA presynaptic complex. The ssDNA-RAD51 filament scans the genome to search for homology and on success, the filament invades the duplex homologous DNA and initiates strand exchange, creating a displacement loop (D-loop). Cancers 2021, 13(9), 2083; https://doi.org/10.3390/cancers13092083 This is an open access article distributed under the Creative Commons Attribution License 2.3. DNA Synthesis The 3′ invading strand primes DNA synthesis through the recruitment of DNA and the copy of the invaded DNA molecule. Numerous polymerases are involved in this process, although Polδ has been proposed to play a primary role. A protein complexes HROB-MCM8–MCM9 and HELQ are proposed to have redundant helicase functions to promote DNA synthesis during HR. Cancers 2021, 13(9), 2083; https://doi.org/10.3390/cancers13092083 This is an open access article distributed under the Creative Commons Attribution License 2.4. Formation and Resolution of HR Intermediates Strand invasion and DNA synthesis lead to the formation of different intermediates whose processing leads to gene conversion either associated with crossover products or not. The invading strand can be disassembled, channeling DSB repair toward synthesis-dependent strand annealing (SDSA). If stabilized, the D-loop can lead to DSB repair by break-induced repair (BIR) or to the formation of double Holliday junctions that can be either dissolved by the BLM- TOP3A-RMI1/2 complex or resolved by the structure-specific resolvases MUS81-EME1, GEN1 or SLX1. Cancers 2021, 13(9), 2083; https://doi.org/10.3390/cancers13092083 This is an open access article distributed under the Creative Commons Attribution License BLM is a RecQ family DNA helicase. It is mutated in Bloom syndrome and plays several roles, sometimes contradictory. For eg. It has been shown to suppress crossovers in mitotic cells while repair mitotic DNA gaps via HR*. BLM is involved in different steps of HR, including end resection at HR initiation, D-loop rejection and double Holliday Junction (dHJ) resolution at HR termination. At resection initiation, depending on the cell cycle phase that modifies its interacting partners, BLM either favors the loading of 53BP1 on the DSB in G1 phase, preventing the initiation of unscheduled resection, or, in contrast, favors resection in S phase when interacting with TOP3. Cancers 2021, 13(9), 2083;https://doi.org/10.3390/cancers13092083 *Genetics. 2017 Nov; 207(3): 923–933. 2.5. Accessory Proteins RAD54, a member of the SWI2/SNF2 protein family (ATP-dependent chromatin remodelers), interacts with RAD51, and in vitro studies have proposed that it functions as a RAD51 cofactor. RAD54 catalyzes the extension of joint molecules and stabilizes the D-loop. A family of six proteins (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3, and RAD51AP1), known as the RAD51 paralogs (i.e., proteins that share sequence homology with RAD51 in a given species), has been identified in mammals. Two distinct complexes have been identified: RAD51B–RAD51C–RAD51D–XRCC2 (BCDX2) and RAD51C–XRCC3 (CX3). RAD51 paralogs favor the recruitment of RAD51 to DNA damage sites and promote the formation and stabilization of the RAD51 nucleoprotein filament. RAD51 paralogs also influence gene conversion tract length. The SWSAP1 protein, a noncanonical paralog of RAD51, forms the so-called SHU complex when associated with SWS1 (SWSAP1-SWS1). SHU interacts with RAD51 and regulates its function. Cancers 2021, 13(9), 2083; https://doi.org/10.3390/cancers13092083 NHEJ Canonical non-homologous end joining (C-NHEJ) pathway. Ku 70/80 C-NHEJ depends on Ku heterodimer and DNA-PK catalytic subunit (DNA-PKcs), which together form the DNA-PK holoenzyme. Ku is a first responder at DSBs DNA-PKcs and provides a docking site for DNA-PKcs. Unlike MRN, which can bind internally, Ku requires a free DNA end for binding and cannot associate with MRN, CtIP, Artemis etc most blocked ends. Several nucleases, including tyrosyl-DNA Lipase 4, XRCC 4, XLF phosphodiesterase 1 and 2 (TDP1/2) and Artemis, can remove hairpins, damaged bases or protein-DNA adducts. The DNA ends are processed by additional enzymes and rejoined by the LIG4/XRCC4/XLF complex Figure Adapted from Transl Cancer Res 2013;2(3):163-177 Front. Cell Dev. Biol., 12 July 2021 | https://doi.org/10.3389/fcell.2021.708763 Corresponding Enzymes in Prokaryotic and Eukaryotic NHEJ Components Annu Rev Biochem. 2010; 79: 181–211.  Ku is an abundant, highly conserved DNA binding protein found in both prokaryotes and eukaryotes that plays essential roles in the maintenance of genome integrity. In eukaryotes, Ku is a heterodimer comprised of two subunits, Ku70 and Ku80, that is best characterized for its central role as the initial DNA end binding factor in the "classical" non- homologous end joining (C-NHEJ) pathway, the main DNA double-strand break (DSB) repair pathway in mammals. Ku binds double-stranded DNA ends with high affinity in a sequence-independent manner through a central ring formed by the intertwined strands of the Ku70 and Ku80 subunits. At the break, Ku directly and indirectly interacts with several C-NHEJ factors and processing enzymes, serving as the scaffold for the entire DNA repair complex. Clark et al. Molecular Biology 2019 Elsevier Inc. General Steps of Nonhomologous DNA End Joining Ku binding to the DNA ends at a DSB improves binding by nuclease, polymerase and ligase components. Flexibility in the loading of these enzymatic components, the option to load repeatedly (iteratively), and independent processing of the two DNA end all permit mechanistic flexibility for the NHEJ process. This mechanistic flexibility is essential to permit NHEJ to handle a very diverse array of DSB end configurations and to join them. In addition to the overall mechanistic flexibility, each component exhibits enzymatic flexibility and multifunctionality. Annu Rev Biochem. 2010; 79: 181–211. In addition to C-NHEJ and HRR, there is a third pathway of DSB processing, functioning on simple end-joining principles, but repairing DSBs in a slower speed than C-NHEJ. This repair pathway is considered to be an alternative form of NHEJ and is frequently abbreviated as A-NHEJ, or simply A-EJ. A-NHEJ or B-NHEJ A-EJ is suppressed by C-NHEJ, and possibly also by HRR, and becomes active only when these standard repair processes fail, globally or locally. Therefor it is considered to be a backup pathway and has been abbreviated as B-NHEJ in many instances Transl Cancer Res 2013;2(3):163-177 Several factors have been implicated in A-EJ and their functional diversity has led to the postulate that there are several sub-pathways in operation, engaging prospectively at each DSB on the basis of as of yet undefined parameters in competition with other repair pathways. Gelot et al., Genes 2015, 6(2), 267-298; https://doi.org/10.3390/genes6020267. CC BY 4.0  A-EJ will engage at DSBs where either C-NHEJ or HRR have attempted processing but somehow failed. Thus, at each DSB where A- EJ engages, factors of either C-NHEJ or HRR, particularly those involved at early steps, will be present when A-EJ takes DSB processing over. Also, it is possible, and even likely, that these factors have already operated at DNA ends and have carried out one or more of the initial steps of C-NHEJ or HRR, which of course alters the state of the substrate presented to A-EJ. Furthermore, the presence of C-NHEJ and HRR factors at the DNA ends may either facilitate or compromise A-EJ. Gelot et al., Genes 2015, 6(2), 267-298; https://doi.org/10.3390/genes6020267. CC BY 4.0 When the engagement of A-EJ follows failure of C-NHEJ, several of the early C-NHEJ factors may be present at the junction, but the process must be abrogated prior to ligation by LIG4. End ligation is carried out with either of the remaining ligases, LIG3 and LIG1. PARP1 is a sensor for DNA discontinuities, originally shown to operate in base excision and single-strand break repair. Previous work implicated PARP1 also in repair by A-EJ. There is even evidence for competition between Ku and PARP1 for DSBs raising the possibility that pre-existing C-NHEJ factors at the DSB compromise A-EJ. DNA end stabilization provided in C-NHEJ by Ku may be provided in A- EJ by histone H1. However, it should be emphasized that to date the evidence for a role of histone H1 in A-EJ is of purely biochemical nature. A-EJ is considered to be a mechanistically distinct repair pathway, and has been shown to be active throughout the cell cycle. It is markedly enhanced in the G2 as compared to G1 phase, and is compromised in stationary-phase cells tested either in the G1 or G2 phase of the cell cycle. There are speculations that the latter response may be regulated by phosphorylation of BRCA1 at S988 through Chk2, where in its phosphorylated form BRCA1 promotes error-free NHEJ and suppresses mutagenic A-EJ. Therewith, it reduces the size of deletions at the breakpoint junction. However, this dependency is more likely in G2 than in G1 cells as BRCA1/CtIP/MRN initiates DSB resection during S/G2 phases, and therefore alternative mechanisms should be explored.  Non Homologous End Joining. It starts with recognition of the DNA ends by the Ku70/80 heterodimer, which recruits DNA-PKcs. If the ends are incompatible, nucleases such as Artemis can trim the ends. The XRCC4-DNA Ligase IV-XLF ligation complex seals the break. Homologous Recombination. The MRN-CtIP- complex starts resection on the breaks to generate single stranded DNA (ssDNA). After resection the break can no longer be repaired by NHEJ. The ssDNA is first coated by RPA, which is subsequently replaced by Rad51 with the help of BRCA2. These Rad51 nucleoprotein filaments mediate strand invasion on the homologous template. Extension of the D-loop and capture of the second end lead to repair. Genome Integrity volume 3, Article number: 9 (2012). This is an Open Access article distributed under the terms of the Creative Commons Attribution License Thankyou