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1.
Proc Natl Acad Sci U S A ; 117(44): 27566-27577, 2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33077594

RESUMO

Recent studies have implicated DNA polymerases θ (Pol θ) and ß (Pol ß) as mediators of alternative nonhomologous end-joining (Alt-NHEJ) events, including chromosomal translocations. Here we identify subunits of the replicative DNA polymerase δ (Pol δ) as promoters of Alt-NHEJ that results in more extensive intrachromosomal mutations at a single double-strand break (DSB) and more frequent translocations between two DSBs. Depletion of the Pol δ accessory subunit POLD2 destabilizes the complex, resulting in degradation of both POLD1 and POLD3 in human cells. POLD2 depletion markedly reduces the frequency of translocations with sequence modifications but does not affect the frequency of translocations with exact joins. Using separation-of-function mutants, we show that both the DNA synthesis and exonuclease activities of the POLD1 subunit contribute to translocations. As described in yeast and unlike Pol θ, Pol δ also promotes homology-directed repair. Codepletion of POLD2 with 53BP1 nearly eliminates translocations. POLD1 and POLD2 each colocalize with phosphorylated H2AX at ionizing radiation-induced DSBs but not with 53BP1. Codepletion of POLD2 with either ligase 3 (LIG3) or ligase 4 (LIG4) does not further reduce translocation frequency compared to POLD2 depletion alone. Together, these data support a model in which Pol δ promotes Alt-NHEJ in human cells at DSBs, including translocations.


Assuntos
Reparo do DNA por Junção de Extremidades , DNA Polimerase III/metabolismo , Translocação Genética , Quebras de DNA de Cadeia Dupla , DNA Polimerase III/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Células HeLa , Humanos , RNA Interferente Pequeno/metabolismo
2.
Mol Cell ; 55(6): 818-828, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25132173

RESUMO

CAG/CTG trinucleotide repeats are unstable, fragile sequences that strongly position nucleosomes, but little is known about chromatin modifications required to prevent genomic instability at these or other structure-forming sequences. We discovered that regulated histone H4 acetylation is required to maintain CAG repeat stability and promote gap-induced sister chromatid recombination. CAG expansions in the absence of H4 HATs NuA4 and Hat1 and HDACs Sir2, Hos2, and Hst1 depended on Rad52, Rad57, and Rad5 and were therefore arising through homology-mediated postreplication repair (PRR) events. H4K12 and H4K16 acetylation were required to prevent Rad5-dependent CAG repeat expansions, and H4K16 acetylation was enriched at CAG repeats during S phase. Genetic experiments placed the RSC chromatin remodeler in the same PRR pathway, and Rsc2 recruitment was coincident with H4K16 acetylation. Here we have utilized a repetitive DNA sequence that induces endogenous DNA damage to identify histone modifications that regulate recombination efficiency and fidelity during postreplication gap repair.


Assuntos
Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Troca de Cromátide Irmã , Repetições de Trinucleotídeos/genética , Acetilação , Montagem e Desmontagem da Cromatina , Cromossomos Fúngicos , Dano ao DNA , Reparo do DNA , Replicação do DNA , Genoma Fúngico , Instabilidade Genômica , Histona Acetiltransferases/genética , Histona Desacetilases/genética , Histonas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
3.
Crit Rev Biochem Mol Biol ; 50(2): 142-67, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25608779

RESUMO

The expansion of repeated sequences is the cause of over 30 inherited genetic diseases, including Huntington disease, myotonic dystrophy (types 1 and 2), fragile X syndrome, many spinocerebellar ataxias, and some cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Repeat expansions are dynamic, and disease inheritance and progression are influenced by the size and the rate of expansion. Thus, an understanding of the various cellular mechanisms that cooperate to control or promote repeat expansions is of interest to human health. In addition, the study of repeat expansion and contraction mechanisms has provided insight into how repair pathways operate in the context of structure-forming DNA, as well as insights into non-canonical roles for repair proteins. Here we review the mechanisms of repeat instability, with a special emphasis on the knowledge gained from the various model systems that have been developed to study this topic. We cover the repair pathways and proteins that operate to maintain genome stability, or in some cases cause instability, and the cross-talk and interactions between them.


Assuntos
Reparo do DNA/genética , DNA/genética , Conformação de Ácido Nucleico , Expansão das Repetições de Trinucleotídeos/genética , Fragilidade Cromossômica/genética , DNA/química , Dano ao DNA , Replicação do DNA/genética , Doenças Genéticas Inatas/classificação , Doenças Genéticas Inatas/etiologia , Instabilidade Genômica , Humanos , Recombinação Genética
4.
PLoS One ; 15(12): e0237759, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33332350

RESUMO

DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in "clustered" oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in "real time" by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.


Assuntos
Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dano ao DNA/genética , Reparo do DNA/genética , DNA/genética , Estresse Oxidativo/genética , Linhagem Celular , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Genoma/genética , Células HEK293 , Humanos , Luz , RNA Guia de Cinetoplastídeos/genética
5.
Cell Rep ; 27(4): 1151-1164.e5, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31018130

RESUMO

Common fragile sites (CFSs) are genomic regions that display gaps and breaks in human metaphase chromosomes under replication stress and are often deleted in cancer cells. We studied an ∼300-bp subregion (Flex1) of human CFS FRA16D in yeast and found that it recapitulates characteristics of CFS fragility in human cells. Flex1 fragility is dependent on the ability of a variable-length AT repeat to form a cruciform structure that stalls replication. Fragility at Flex1 is initiated by structure-specific endonuclease Mus81-Mms4 acting together with the Slx1-4/Rad1-10 complex, whereas Yen1 protects Flex1 against breakage. Sae2 is required for healing of Flex1 after breakage. Our study shows that breakage within a CFS can be initiated by nuclease cleavage at forks stalled at DNA structures. Furthermore, our results suggest that CFSs are not just prone to breakage but also are impaired in their ability to heal, and this deleterious combination accounts for their fragility.


Assuntos
Quebra Cromossômica , Sítios Frágeis do Cromossomo/genética , Cromossomos Humanos Par 16/genética , Replicação do DNA , Endonucleases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Humanos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequências de Repetição em Tandem
6.
Genetics ; 208(3): 963-976, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29305386

RESUMO

CAG/CTG trinucleotide repeats are unstable sequences that are difficult to replicate, repair, and transcribe due to their structure-forming nature. CAG repeats strongly position nucleosomes; however, little is known about the chromatin remodeling needed to prevent repeat instability. In a Saccharomyces cerevisiae model system with CAG repeats carried on a YAC, we discovered that the chromatin remodeler Isw1 is required to prevent CAG repeat expansions during transcription. CAG repeat expansions in the absence of Isw1 were dependent on both transcription-coupled repair (TCR) and base-excision repair (BER). Furthermore, isw1∆ mutants are sensitive to methyl methanesulfonate (MMS) and exhibit synergistic MMS sensitivity when combined with BER or TCR pathway mutants. We conclude that CAG expansions in the isw1∆ mutant occur during a transcription-coupled excision repair process that involves both TCR and BER pathways. We observed increased RNA polymerase II (RNAPII) occupancy at the CAG repeat when transcription of the repeat was induced, but RNAPII binding did not change in isw1∆ mutants, ruling out a role for Isw1 remodeling in RNAPII progression. However, nucleosome occupancy over a transcribed CAG tract was altered in isw1∆ mutants. Based on the known role of Isw1 in the reestablishment of nucleosomal spacing after transcription, we suggest that a defect in this function allows DNA structures to form within repetitive DNA tracts, resulting in inappropriate excision repair and repeat-length changes. These results establish a new function for Isw1 in directly maintaining the chromatin structure at the CAG repeat, thereby limiting expansions that can occur during transcription-coupled excision repair.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Expansão das Repetições de Trinucleotídeos , Rearranjo Gênico , Repetições de Trinucleotídeos
7.
DNA Repair (Amst) ; 56: 156-165, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28641941

RESUMO

Eukaryotic genomes contain many repetitive DNA sequences that exhibit size instability. Some repeat elements have the added complication of being able to form secondary structures, such as hairpin loops, slipped DNA, triplex DNA or G-quadruplexes. Especially when repeat sequences are long, these DNA structures can form a significant impediment to DNA replication and repair, leading to DNA nicks, gaps, and breaks. In turn, repair or replication fork restart attempts within the repeat DNA can lead to addition or removal of repeat elements, which can sometimes lead to disease. One important DNA repair mechanism to maintain genomic integrity is recombination. Though early studies dismissed recombination as a mechanism driving repeat expansion and instability, recent results indicate that mitotic recombination is a key pathway operating within repetitive DNA. The action is two-fold: first, it is an important mechanism to repair nicks, gaps, breaks, or stalled forks to prevent chromosome fragility and protect cell health; second, recombination can cause repeat expansions or contractions, which can be deleterious. In this review, we summarize recent developments that illuminate the role of recombination in maintaining genome stability at DNA repeats.


Assuntos
Expansão das Repetições de DNA , Replicação do DNA , DNA/metabolismo , Recombinação Genética , Animais , DNA/química , Reparo do DNA , Instabilidade Genômica , Humanos , Conformação de Ácido Nucleico , Leveduras/genética
8.
Front Genet ; 5: 296, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25250043

RESUMO

DNA repair must take place in the context of chromatin, and chromatin modifications and DNA repair are intimately linked. The study of double-strand break repair has revealed numerous histone modifications that occur after induction of a DSB, and modification of the repair factors themselves can also occur. In some cases the function of the modification is at least partially understood, but in many cases it is not yet clear. Although DSB repair is a crucial activity for cell survival, DSBs account for only a small percentage of the DNA lesions that occur over the lifetime of a cell. Repair of single-strand gaps, nicks, stalled forks, alternative DNA structures, and base lesions must also occur in a chromatin context. There is increasing evidence that these repair pathways are also regulated by histone modifications and chromatin remodeling. In this review, we will summarize the current state of knowledge of chromatin modifications that occur during non-DSB repair, highlighting similarities and differences to DSB repair as well as remaining questions.

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