43 research outputs found
Cooperation of DNA-PKcs and WRN helicase in the maintenance of telomeric D-loops
Werner syndrome
is an inherited human progeriod syndrome caused by mutations in the gene
encoding the Werner Syndrome protein, WRN. It has both 3'-5' DNA
helicase and exonuclease activities, and is
suggested to have roles in many aspects of DNA metabolism, including DNA
repair and telomere maintenance. The DNA-PK complex also functions in both
DNA double strand break repair and telomere maintenance. Interaction
between WRN and the DNA-PK complex has been reported in DNA double strand
break repair, but their possible cooperation at telomeres has not been
reported. This study analyzes thein vitro and in vivo
interaction at the telomere between WRN and DNA-PKcs, the catalytic subunit
of DNA-PK. The results show that DNA-PKcs selectively stimulates WRN
helicase but not WRN exonuclease in vitro, affecting that WRN
helicase unwinds and promotes the release of the full-length invading strand
of a telomere D-loop model substrate. In addition, the length of telomeric
G-tails decreases in DNA-PKcs knockdown cells, and this phenotype is
reversed by overexpression of WRN helicase. These results suggest that WRN
and DNA-PKcs may cooperatively prevent G-tail shortening in vivo
Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks
WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and −467 in WRN as major phosphorylation sites mediated by DNA-PK. In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and −467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair
Werner Protein Cooperates with the XRCC4-DNA Ligase IV Complex in End-Processing †
Werner syndrome is a rare human disease characterized by the premature onset of aging-associated pathologies, cancer predisposition, and genomic instability. The Werner protein (WRN), which is defective in Werner syndrome (WS) patients, belongs to the RecQ family helicases and interacts with several DNA metabolic proteins, including DNA repair factors and telomere associated proteins. Nonhomologous end-joining (NHEJ) is an important pathway in the repair of DNA double strand breaks (DSBs), and the DNA-PK complex, composed of the heterodimer Ku 70/86 and the DNA-PK catalytic subunit (DNA-PKcs), together with the XRCC4-DNA ligase IV complex (X4L4), are major factors. One of the most prominent protein interactions of WRN is with Ku 70/86, and it is possible that WRN is involved in NHEJ via its associations with Ku 70/86 and DNA-PKcs. This study demonstrates that WRN physically interacts with the major NHEJ factor, X4L4, which stimulates WRN exonuclease but not its helicase activity. The human RecQ helicase, BLM, which possesses only helicase activity, does not bind to X4L4, and its helicase activity is not affected by X4L4. In a DNA end-joining assay, we find that a substrate, which is processed by WRN, is ligated by X4L4, thus further supporting the significance of their functional interaction
SIRT6 stabilizes DNA-dependent Protein Kinase at chromatin for DNA double-strand break repair
The Sir2 chromatin regulatory factor links maintenance
of genomic stability to life span extension in yeast. The mammalian Sir2
family member SIRT6 has been proposed to have analogous functions, because
SIRT6-deficiency leads to shortened life span and an aging-like
degenerative phenotype in mice, and SIRT6 knockout cells exhibit genomic
instability and DNA damage hypersensitivity. However, the molecular mechanisms
underlying these defects are not fully understood. Here, we show that
SIRT6 forms a macromolecular complex with the DNA double-strand break (DSB)
repair factor DNA-PK (DNA-dependent protein kinase) and promotes DNA DSB
repair. In response to DSBs, SIRT6 associates dynamically with chromatin
and is necessary for an acute decrease in global cellular acetylation
levels on histone H3 Lysine 9. Moreover, SIRT6 is required for
mobilization of the DNA-PK catalytic subunit (DNA-PKcs) to chromatin in response
to DNA damage and stabilizes DNA-PKcs at chromatin adjacent to an induced
site-specific DSB. Abrogation of these SIRT6 activities leads to impaired
resolution of DSBs. Together, these findings elucidate a mechanism whereby
regulation of dynamic interaction of a DNA repair factor with chromatin
impacts on the efficiency of repair, and establish a link between chromatin
regulation, DNA repair, and a mammalian Sir2 factor
Acetylation Regulates WRN Catalytic Activities and Affects Base Excision DNA Repair
Background: The Werner protein (WRN), defective in the premature aging disorder Werner syndrome, participates in a number of DNA metabolic processes, and we have been interested in the possible regulation of its function in DNA repair by post-translational modifications. Acetylation mediated by histone acetyltransferases is of key interest because of its potential importance in aging, DNA repair and transcription. Methodology/Principal Findings: Here, we have investigated the p300 acetylation mediated changes on the function of WRN in base excision DNA repair (BER). We show that acetylation of WRN increases in cells treated with methyl methanesulfonate (MMS), suggesting that acetylation of WRN may play a role in response to DNA damage. This hypothesis is consistent with our findings that acetylation of WRN stimulates its catalytic activities in vitro and in vivo, and that acetylated WRN enhances pol b-mediated strand displacement DNA synthesis more than unacetylated WRN. Furthermore, we show that cellular exposure to the histone deacetylase inhibitor sodium butyrate stimulates long patch BER in wild type cells but not in WRN depleted cells, suggesting that acetylated WRN participates significantly in this process. Conclusion/Significance: Collectively, these results provide the first evidence for a specific role of p300 mediated WRN acetylation in regulating its function during BER
Mechanisms of accurate translesion synthesis by human DNA polymerase η
The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase η (pol η), which is involved in the replication of damaged DNA. Pol η catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol η can catalyze translesion synthesis past an abasic (AP) site analog, N-2-acetylaminofluorene (AAF)-modified guanine, and a cisplatin-induced intrastrand cross-link between two guanines. Pol η preferentially incorporated dAMP and dGMP opposite AP, and dCMP opposite AAF-G and cisplatin-GG, but other nucleotides were also incorporated opposite these lesions. However, after incorporating an incorrect nucleotide opposite a lesion, pol η could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol η could continue chain elongation, whereas pol α could not. Thus, the fidelity of translesion synthesis by human pol η relies not only on the ability of this enzyme to incorporate the correct nucleotide opposite a lesion, but also on its ability to elongate only DNA chains that have a correctly incorporated nucleotide opposite a lesion
Genomic Instability and Cancer Risk Associated with Erroneous DNA Repair
Many cancers develop as a consequence of genomic instability, which induces genomic rearrangements and nucleotide mutations. Failure to correct DNA damage in DNA repair defective cells, such as in BRCA1 and BRCA2 mutated backgrounds, is directly associated with increased cancer risk. Genomic rearrangement is generally a consequence of erroneous repair of DNA double-strand breaks (DSBs), though paradoxically, many cancers develop in the absence of DNA repair defects. DNA repair systems are essential for cell survival, and in cancers deficient in one repair pathway, other pathways can become upregulated. In this review, we examine the current literature on genomic alterations in cancer cells and the association between these alterations and DNA repair pathway inactivation and upregulation