scholarly journals Homology Search and Choice of Homologous Partner during Mitotic Recombination

1999 ◽  
Vol 19 (6) ◽  
pp. 4134-4142 ◽  
Author(s):  
Ori Inbar ◽  
Martin Kupiec
Keyword(s):  
Homologous Recombination ◽  
Dna Sequences ◽  
Sequence Divergence ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Repeated Sequences ◽  
Homology Search ◽  
Strand Breaks ◽  
Repair Genes

ABSTRACT Homologous recombination is an important DNA repair mechanism in vegetative cells. During the repair of double-strand breaks, genetic information is transferred between the interacting DNA sequences (gene conversion). This event is often accompanied by a reciprocal exchange between the homologous molecules, resulting in crossing over. The repair of DNA damage by homologous recombination with repeated sequences dispersed throughout the genome might result in chromosomal aberrations or in the inactivation of genes. It is therefore important to understand how the suitable homologous partner for recombination is chosen. We have developed a system in the yeast Saccharomyces cerevisiae that can monitor the fate of a chromosomal double-strand break without the need to select for recombinants. The broken chromosome is efficiently repaired by recombination with one of two potential partners located elsewhere in the genome. One of the partners has homology to the broken ends of the chromosome, whereas the other is homologous to sequences distant from the break. Surprisingly, a large proportion of the repair is carried out by recombination involving the sequences distant from the broken ends. This repair is very efficient, despite the fact that it requires the processing of a large chromosomal region flanking the break. Our results imply that the homology search involves extensive regions of the broken chromosome and is not carried out exclusively by sequences adjacent to the double-strand break. We show that the mechanism that governs the choice of homologous partners is affected by the length and sequence divergence of the interacting partners, as well as by mutations in the mismatch repair genes. We present a model to explain how the suitable homologous partner is chosen during recombinational repair. The model provides a mechanism that may guard the integrity of the genome by preventing recombination between dispersed repeated sequences.

332 COMBINATION OF ZINC FINGER NUCLEASES AND MODIFIED BACTERIAL ARTIFICIAL CHROMOSOMES REVEALS TREMENDOUS RATES OF HOMOLOGOUS RECOMBINATION

2013 ◽  
Vol 25 (1) ◽  
pp. 314
Author(s):  
P. Fezert ◽  
A. Wuensch ◽  
E. Wolf ◽  
N. Klymiuk
Keyword(s):  
Homologous Recombination ◽  
Zinc Finger ◽  
Reporter Gene ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Zinc Finger Nucleases ◽  
Strand Breaks ◽  
Artificial Chromosomes ◽  
Targeting Vector

DNA-based vectors have been used for decades to modify the genomes of mammalian cells by homologous recombination in a specific and site-directed way. Even though various modifications of the procedure have been presented, efficiency is relatively low for many target sites and novel projects still have an unforeseeable outcome. This is in particularly true for site-directed mutagenesis in primary cells intended for use in the generation of large animal models because of their impaired predisposition for homologous recombination compared with stem cells. The recent development of site-specific nucleases is based on a completely different principle: they do not necessarily involve recombination between DNA strands, but rather make use of the inefficient correction of double-strand breaks in the genomic DNA by the cellular DNA repair machinery after such a double-strand break has been introduced by a synthetic enzyme that directed nuclease activity to a defined site in the genome. Here, we intended to evaluate the potential of zinc finger nucleases (ZFN) to introduce a lacZ reporter gene into the CFTR locus. Initially, the efficiency of 3 different ZFN pairs was examined under different conditions revealing modification efficiencies between 0 and 38%. An optimized protocol was used to combine the most efficient ZFN pair with either a bacterial artificial chromosome (BAC) vector or a conventional targeting vector carrying the desired modification. Although the conventional vector failed to introduce the reporter gene in any of more than 200 clones examined, the BAC correctly modified the target site in 32 of 75 clones in a heterozygous way and in 10 out of 75 clones in a homozygous way. However, the introduction of small vector fragments into the CFTR locus in rare cases indicated that the ZFN caused a double-strand break but the vector was not able to act as a recombination donor. On the other hand, transfection of the BAC alone only resulted in 1 modified clone out of 98 and, thus, our data strongly support the hypothesis that the forced introduction of double-strand breaks dramatically increases the rate of homologous recombination, but they also provide indication that the design of the targeting vector has a profound influence on the efficiency.

scholarly journals Role of RAD52 Epistasis Group Genes in Homologous Recombination and Double-Strand Break Repair

2002 ◽  
Vol 66 (4) ◽  
pp. 630-670 ◽  
Author(s):  
Lorraine S. Symington
Keyword(s):  
Homologous Recombination ◽  
Ionizing Radiation ◽  
Strand Break ◽  
Double Strand Break ◽  
Dna Lesions ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Repair Pathway ◽  
Strand Breaks ◽  
Break Repair

SUMMARY The process of homologous recombination is a major DNA repair pathway that operates on DNA double-strand breaks, and possibly other kinds of DNA lesions, to promote error-free repair. Central to the process of homologous recombination are the RAD52 group genes (RAD50, RAD51, RAD52, RAD54, RDH54/TID1, RAD55, RAD57, RAD59, MRE11, and XRS2), most of which were identified by their requirement for the repair of ionizing-radiation-induced DNA damage in Saccharomyces cerevisiae. The Rad52 group proteins are highly conserved among eukaryotes, and Rad51, Mre11, and Rad50 are also conserved in prokaryotes and archaea. Recent studies showing defects in homologous recombination and double-strand break repair in several human cancer-prone syndromes have emphasized the importance of this repair pathway in maintaining genome integrity. Although sensitivity to ionizing radiation is a universal feature of rad52 group mutants, the mutants show considerable heterogeneity in different assays for recombinational repair of double-strand breaks and spontaneous mitotic recombination. Herein, I provide an overview of recent biochemical and structural analyses of the Rad52 group proteins and discuss how this information can be incorporated into genetic studies of recombination.

scholarly journals PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks

2020 ◽  
Vol 48 (9) ◽  
pp. 4915-4927 ◽  
Author(s):  
Ignacio Alonso-de Vega ◽  
Maria Cristina Paz-Cabrera ◽  
Magdalena B Rother ◽  
Wouter W Wiegant ◽  
Cintia Checa-Rodríguez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Focus Formation ◽  
Strand Breaks ◽  
Break Repair

Abstract Post-translational histone modifications and chromatin remodelling play a critical role controlling the integrity of the genome. Here, we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating DNA damage-induced focus formation of 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knockdown leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knockdown decreases CtIP and BRCA1 protein and mRNA levels, an effect that is dependent on the demethylase activity of PHF2. Furthermore, PHF2-depleted cells display genome instability and are mildly sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.

scholarly journals PHF2 regulates homology-directed DNA repair by controlling the resection of DNA double strand breaks

2019 ◽  
Author(s):  
Ignacio Alonso-de Vega ◽  
M. Cristina Paz-Cabrera ◽  
Wouter W. Wiegant ◽  
Cintia Checa-Rodríguez ◽  
Pablo Huertas ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Double Strand Break Repair ◽  
Focus Formation ◽  
Strand Breaks ◽  
Knock Down

ABSTRACTPost-translational histone modifications and chromatin remodelling play a critical role in the mechanisms controlling the integrity of the genome. Here we identify histone lysine demethylase PHF2 as a novel regulator of the DNA damage response by regulating the balance between DNA damage-induced focus formation by 53BP1 and BRCA1, critical factors in the pathway choice for DNA double strand break repair. PHF2 knock down leads to impaired BRCA1 focus formation and delays the resolution of 53BP1 foci. Moreover, irradiation-induced RPA phosphorylation and focus formation, as well as localization of CtIP, required for DNA end resection, to sites of DNA lesions are affected by depletion of PHF2. These results are indicative of a defective resection of double strand breaks and thereby an impaired homologous recombination upon PHF2 depletion. In accordance with these data, Rad51 focus formation and homology-directed double strand break repair is inhibited in cells depleted for PHF2. Importantly, we demonstrate that PHF2 knock down decreases CtIP and BRCA1 protein and mRNA levels and cells depleted of PHF2 display genome instability and are sensitive to the inhibition of PARP. Together these results demonstrate that PHF2 promotes DNA repair by homologous recombination by controlling CtIP-dependent resection of double strand breaks.

scholarly journals End-resection at DNA double-strand breaks in the three domains of life

2013 ◽  
Vol 41 (1) ◽  
pp. 314-320 ◽  
Author(s):  
John K. Blackwood ◽  
Neil J. Rzechorzek ◽  
Sian M. Bray ◽  
Joseph D. Maman ◽  
Luca Pellegrini ◽  
...  
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Homologous Chromosomes ◽  
Domains Of Life ◽  
Strand Invasion ◽  
End Resection

During DNA repair by HR (homologous recombination), the ends of a DNA DSB (double-strand break) must be resected to generate single-stranded tails, which are required for strand invasion and exchange with homologous chromosomes. This 5′–3′ end-resection of the DNA duplex is an essential process, conserved across all three domains of life: the bacteria, eukaryota and archaea. In the present review, we examine the numerous and redundant helicase and nuclease systems that function as the enzymatic analogues for this crucial process in the three major phylogenetic divisions.

Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum

1988 ◽  
Vol 8 (7) ◽  
pp. 2779-2786
Author(s):  
K S Katz ◽  
D I Ratner
Keyword(s):  
Homologous Recombination ◽  
Dictyostelium Discoideum ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Restriction Endonucleases ◽  
Linear Molecule ◽  
Strand Breaks ◽  
Transformation Vector ◽  
Closed Circle ◽  
Primary Vector

We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.

scholarly journals Single-molecule live-cell imaging reveals RecB-dependent function of DNA polymerase IV in double strand break repair

2020 ◽  
Vol 48 (15) ◽  
pp. 8490-8508 ◽  
Author(s):  
Sarah S Henrikus ◽  
Camille Henry ◽  
Amy E McGrath ◽  
Slobodan Jergic ◽  
John P McDonald ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Single Molecule ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
Pol Iv ◽  
Dna Polymerase Iv

Abstract Several functions have been proposed for the Escherichia coli DNA polymerase IV (pol IV). Although much research has focused on a potential role for pol IV in assisting pol III replisomes in the bypass of lesions, pol IV is rarely found at the replication fork in vivo. Pol IV is expressed at increased levels in E. coli cells exposed to exogenous DNA damaging agents, including many commonly used antibiotics. Here we present live-cell single-molecule microscopy measurements indicating that double-strand breaks induced by antibiotics strongly stimulate pol IV activity. Exposure to the antibiotics ciprofloxacin and trimethoprim leads to the formation of double strand breaks in E. coli cells. RecA and pol IV foci increase after treatment and exhibit strong colocalization. The induction of the SOS response, the appearance of RecA foci, the appearance of pol IV foci and RecA-pol IV colocalization are all dependent on RecB function. The positioning of pol IV foci likely reflects a physical interaction with the RecA* nucleoprotein filaments that has been detected previously in vitro. Our observations provide an in vivo substantiation of a direct role for pol IV in double strand break repair in cells treated with double strand break-inducing antibiotics.

scholarly journals Effects of Mutations in DNA Repair Genes on Formation of Ribosomal DNA Circles and Life Span inSaccharomyces cerevisiae

1999 ◽  
Vol 19 (5) ◽  
pp. 3848-3856 ◽  
Author(s):  
Peter U. Park ◽  
Pierre-Antoine Defossez ◽  
Leonard Guarente
Keyword(s):  
Dna Repair ◽  
Homologous Recombination ◽  
Life Span ◽  
Ribosomal Dna ◽  
Initial Step ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Mother Cells ◽  
Mutant Cells ◽  
Repair Genes

ABSTRACT A cause of aging in Saccharomyces cerevisiae is the accumulation of extrachromosomal ribosomal DNA circles (ERCs). Introduction of an ERC into young mother cells shortens life span and accelerates the onset of age-associated sterility. It is important to understand the process by which ERCs are generated. Here, we demonstrate that homologous recombination is necessary for ERC formation. rad52 mutant cells, defective in DNA repair through homologous recombination, do not accumulate ERCs with age, and mutations in other genes of the RAD52 class have varying effects on ERC formation. rad52 mutation leads to a progressive delocalization of Sir3p from telomeres to other nuclear sites with age and, surprisingly, shortens life span. We speculate that spontaneous DNA damage, perhaps double-strand breaks, causes lethality in mutants of the RAD52 class and may be an initial step of aging in wild-type cells.

Double-strand-break-induced homologous recombination in mammalian cells

2001 ◽  
Vol 29 (2) ◽  
pp. 196-201 ◽  
Author(s):  
R. D. Johnson ◽  
M. Jasin
Keyword(s):  
Homologous Recombination ◽  
Sister Chromatid ◽  
Mammalian Cells ◽  
Indirect Evidence ◽  
Strand Break ◽  
Double Strand Breaks ◽  
Crossing Over ◽  
Dna Double Strand Breaks ◽  
Dsb Repair ◽  
Strand Breaks

In mammalian cells, the repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. Indirect evidence, including that from gene targeting and random integration experiments, had suggested that non-homologous mechanisms were significantly more frequent than homologous ones. However, more recent experiments indicate that homologous recombination is also a prominent DSB repair pathway. These experiments show that mammalian cells use homologous sequences located at multiple positions throughout the genome to repair a DSB. However, template preference appears to be biased, with the sister chromatid being preferred by 2–3 orders of magnitude over a homologous or heterologous chromosome. The outcome of homologous recombination in mammalian cells is predominantly gene conversion that is not associated with crossing-over. The preference for the sister chromatid and the bias against crossing-over seen in mitotic mammalian cells may have developed in order to reduce the potential for genome alterations that could occur when other homologous repair templates are utilized. In attempts to understand further the mechanism of homologous recombination, the proteins that promote this process are beginning to be identified. To date, four mammalian proteins have been demonstrated conclusively to be involved in DSB repair by homologous recombination: Rad54, XRCC2, XRCC3 and BRCAI. This paper summarizes results from a number of recent studies.

Sign in / Sign up

Export Citation Format

Share Document