scholarly journals Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

10.3791/2002 ◽  
2010 ◽  
Author(s):  
Andrei Seluanov ◽  
Zhiyong Mao ◽  
Vera Gorbunova
Keyword(s):  
Mammalian Cells ◽  
Strand Break ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Dna Double Strand Break

scholarly journals Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair

2020 ◽  
Vol 61 (5) ◽  
pp. 718-726 ◽  
Author(s):  
Atsushi Shibata ◽  
Penny A Jeggo
Keyword(s):  
Mammalian Cells ◽  
Binding Capacity ◽  
Susceptibility Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Breast Cancer Susceptibility Gene ◽  
Dsb Repair ◽  
Fast Kinetics ◽  
Dna Double Strand Break ◽  
Slow Kinetics

Abstract p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1’s breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

scholarly journals The Roles of Bacterial DNA Double-Strand Break Repair Proteins in Chromosomal DNA Replication

2020 ◽  
Vol 44 (3) ◽  
pp. 351-368 ◽  
Author(s):  
Anurag Kumar Sinha ◽  
Christophe Possoz ◽  
David R F Leach
Keyword(s):  
Dna Replication ◽  
Cell Viability ◽  
Strand Break ◽  
Double Strand Break ◽  
Replication Initiation ◽  
Genome Replication ◽  
Chromosomal Dna ◽  
Dsb Repair ◽  
Bacterial Dna ◽  
Dna Double Strand Break

ABSTRACT It is well established that DNA double-strand break (DSB) repair is required to underpin chromosomal DNA replication. Because DNA replication forks are prone to breakage, faithful DSB repair and correct replication fork restart are critically important. Cells, where the proteins required for DSB repair are absent or altered, display characteristic disturbances to genome replication. In this review, we analyze how bacterial DNA replication is perturbed in DSB repair mutant strains and explore the consequences of these perturbations for bacterial chromosome segregation and cell viability. Importantly, we look at how DNA replication and DSB repair processes are implicated in the striking recent observations of DNA amplification and DNA loss in the chromosome terminus of various mutant Escherichia coli strains. We also address the mutant conditions required for the remarkable ability to copy the entire E. coli genome, and to maintain cell viability, even in the absence of replication initiation from oriC, the unique origin of DNA replication in wild type cells. Furthermore, we discuss the models that have been proposed to explain these phenomena and assess how these models fit with the observed data, provide new insights and enhance our understanding of chromosomal replication and termination in bacteria.

The power of DNA double‐strand break (DSB) repair testing to predict breast cancer susceptibility

2012 ◽  
Vol 26 (5) ◽  
pp. 2094-2104 ◽  
Author(s):  
Marlen Keimling ◽  
Miriam Deniz ◽  
Dominic Varga ◽  
Andreea Stahl ◽  
Hubert Schrezenmeier ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Susceptibility ◽  
Strand Break ◽  
Breast Cancer Susceptibility ◽  
Double Strand Break ◽  
Dsb Repair ◽  
Dna Double Strand Break

scholarly journals RAD-51-Dependent and -Independent Roles of a Caenorhabditis elegans BRCA2-Related Protein during DNA Double-Strand Break Repair

2005 ◽  
Vol 25 (8) ◽  
pp. 3127-3139 ◽  
Author(s):  
Julie S. Martin ◽  
Nicole Winkelmann ◽  
Mark I. R. Petalcorin ◽  
Michael J. McIlwraith ◽  
Simon J. Boulton
Keyword(s):  
Caenorhabditis Elegans ◽  
Strand Break ◽  
Double Strand Break ◽  
Nonhomologous End Joining ◽  
Related Protein ◽  
End Joining ◽  
Dsb Repair ◽  
Phenotypic Differences ◽  
Dna Double Strand Break ◽  
Radiation Induced

ABSTRACT The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.

Lack of Functional DNA-PKcs and DNA Double-Strand Break Repair Leads to a Repopulating Stem Cell Defect in Scid Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2684-2684
Author(s):  
Youngji Park ◽  
Yuan Lin ◽  
Stanton L. Gerson
Keyword(s):  
Dna Repair ◽  
Stem Cell ◽  
Genomic Stability ◽  
Strand Break ◽  
Double Strand Break ◽  
Scid Mice ◽  
Double Strand Break Repair ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Scid Cells

Abstract Intact function of DNA repair gene is required for maintenance of genomic stability and long term survival of stem cells. We hypothesize that DNA-PKcs, a key factor for DNA double-strand break (DSB) repair, is critical for hematopoietic stem cell (HSC) function. Expression level of DNA-PKcs mRNA monitored by RT-PCR was high in kit+lin− and sca+lin− cells, low in sca+kit+lin− cells and not seen in lin+ cells, implying its role in highly proliferative progenitors. To assess the function of HSCs deficient in DSB repair, serial transplantation capacity of scid (DNA-PKcs−/−) BM cells into lethally irradiated recipients was compared to wildtype BM. Primary transplants of scid BM died after treatment with 2Gy irradiation 4 wks post-transplantation (n=3). In contrast, parental scid mice survived 3Gy irradiation, implying radiation hypersensitivity of scid BM cells after transplantation. No changes were found in the telomere length, cell cycle distribution and apoptosis between the wildtype and scid BM cells after primary transplantation. Scid BM cells failed to repopulate recipients after the third round of transplantation (n=8). To assess competitive repopulating capacity, mixtures of wildtype and scid cells were transplanted into lethally irradiated recipients. BM CFU of primary recipients were predominantly wildtype (8 mice for C3H background, total CFU=262; 5 mice for C56B/6 background, total CFU=336; n>15 per mouse). Scid cells with two independent genetic backgrounds caused consistent repopulation defects, confirming repopulation defect is caused by DNA-PKcs deficiency. All five primary recipients with C56B/6 background was repopulated predominantly by wildtype CFU (wt CFU 93±5% vs. wt CFU of input; 60±31%, p<10−4). Six of eight primary recipients with C3H background had BM cells repopulated by wildtype CFUs (wt CFU 93±9 % vs. wt CFU of input; 65+13 %, p<10−4), and two of eight primary recipients (wt CFU 67±10 %, p>0.05) had BM cells repopulated similar to donor mixture of wildtype and scid. BM cells of all eight primary recipient mice with C3H background were transplanted into secondary recipients. In all cases, including recipients of the primary cells with the mixed chimera, most BM CFU of secondary recipients originated from wildtype (wt CFU 96±7.8 %, total 16 mice, total CFU=511, and CFU=192 from the mixed chimera). Sca+kit+lin− cells were isolated from the secondary recipients, cultured for 2wks and genotyped. All sca+kit+lin− cells were originated from wildtype (total n=73, 6 mice), implying DNA-PKcs function for HSC proliferation. This confirmed that primary recipients had reconstituted with 100% wildtype HSCs and that the mixed chimera reverted to 100% wildtype. Frequency of sca+kit+lin− cells in scid BM was significantly higher than wildtype (scid 1.94±0.5x10−4, n=4 vs. wt 0.92±0.4x10−4, n=4; p=0.017). Frequency of sca+kit+lin− cells in scid secondary recipients became similar to wildtype secondary recipients (scid 0.61±0.2x10−4, n=4 vs. wt 0.48±0.02x10−4, n=3; p=0.25), implying decreased self-renewal of scid HSCs during repetitive transplantation. This indicates that deficiency in DNA double-strand break repair (scid) leads to HSC failure during repetitive transplantation. Thus, intact DNA repair is essential for maintenance and genomic stability of HSCs.

scholarly journals The deSUMOylase SENP2 coordinates homologous recombination and non-homologous end joining by independent mechanisms

2018 ◽  
Author(s):  
Alexander J. Garvin ◽  
Alexandra K. Walker ◽  
Ruth M. Densham ◽  
Anoop Singh Chauhan ◽  
Helen R. Stone ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
End Joining ◽  
Dsb Repair ◽  
Dna Double Strand Break ◽  
Non Homologous End Joining

AbstractSUMOylation in the DNA double-strand break (DSB) response regulates recruitment, activity and clearance of repair factors. However, our understanding of a role for deSUMOylation in this process is limited. Here we identify different mechanistic roles for deSUMOylation in homologous recombination (HR) and non-homologous enjoining (NHEJ) through the investigation of the deSUMOylase SENP2. We find regulated deSUMOylation of MDC1 prevents excessive SUMOylation and its RNF4-VCP mediated clearance from DSBs, thereby promoting NHEJ. In contrast we show HR is differentially sensitive to SUMO availability and SENP2 activity is needed to provide SUMO. SENP2 is amplified as part of the chromosome 3q amplification in many cancers. Increased SENP2 expression prolongs MDC1 foci retention and increases NHEJ and radioresistance. Collectively our data reveal that deSUMOylation differentially primes cells for responding to DSBs and demonstrates the ability of SENP2 to tune DSB repair responses.

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