Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA.
Molecular and Cellular Biology (Impact Factor: 4.78). 12/2004; 24(21):9305-16. DOI: 10.1128/MCB.24.21.9305-9316.2004
Source: PubMed


Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52(-)(/)(-) mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

Download full-text


Available from: Albert Pastink, Mar 17, 2014
  • Source
    • "Cite this article as Cold Spring Harb Perspect Biol 2015;7:a016535 sable for RAD51 focus formation (van Veelen et al. 2005), the synthetic lethality of rad52 mutants with mutants of PALB2, BRCA2, and the RAD51 paralogs RAD51BCD-XRCC2/3 (Fujimori et al. 2001; Feng et al. 2011; Chun et al. 2013; Lok et al. 2013), indicates a role for RAD52 during HR in higher eukaryotes (Rijkers et al. 1998; Stark et al. 2004). Together with Rad52, the Rad51 paralogs Rad55-Rad57 promote the formation of Rad51 filaments and themselves require Rad51 for focus formation (Sung 1997b; Lisby et al. 2004). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Homologous recombination provides high-fidelity DNA repair throughout all domains of life. Live cell fluorescence microscopy offers the opportunity to image individual recombination events in real time providing insight into the in vivo biochemistry of the involved proteins and DNA molecules as well as the cellular organization of the process of homologous recombination. Herein we review the cell biological aspects of mitotic homologous recombination with a focus on Saccharomyces cerevisiae and mammalian cells, but will also draw on findings from other experimental systems. Key topics of this review include the stoichiometry and dynamics of recombination complexes in vivo, the choreography of assembly and disassembly of recombination proteins at sites of DNA damage, the mobilization of damaged DNA during homology search, and the functional compartmentalization of the nucleus with respect to capacity of homologous recombination. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
    Cold Spring Harbor perspectives in biology 03/2015; 7(3). DOI:10.1101/cshperspect.a016535 · 8.68 Impact Factor
  • Source
    • "We have evaluated two distinct HR reporters, DR-GFP (described above, Figure 1A), and SA-GFP, in which HR repair restores a GFP expression cassette via single-strand annealing (SSA). While HDR involves RAD51-dependent gene conversion, SSA is a RAD51-independent HR event resulting in a deletion between two repeats (40). Both repair events likely require end resection and are promoted by BRCA1 (40). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.
    Nucleic Acids Research 05/2014; 42(12). DOI:10.1093/nar/gku421 · 9.11 Impact Factor
  • Source
    • "When a new set of gene-editing reagents is developed for a custom application, the activity levels of nucleases and the frequency of the desired gene-editing event at the target locus must be determined and often need to be optimized for the specific cell type and system being used. This need has previously been met by a variety of methods including gel-based assays to measure mutagenic NHEJ (Guschin et al., 2010), gene addition of fluorescent reporters to measure HDR (Porteus and Baltimore, 2003; Stark et al., 2004), analysis of large numbers of single-cell clones, and the use of optimization assays to measure NHEJ and HDR at engineered reporter loci (Certo et al., 2011). Although each of these assays have their utility, each have important limitations including a lack of sensitivity required for difficult applications (gel-based assays), the use of an indirect rather than a direct measure of genome editing (targeted gene addition of fluorescent reporters), and the need to generate reporter cell lines (Traffic Light Reporter system). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used.
    Cell Reports 03/2014; 7(1). DOI:10.1016/j.celrep.2014.02.040 · 8.36 Impact Factor
Show more