RECQL5 cooperates with Topoisomerase II alpha in DNA decatenation and cell cycle progression.

DNA decatenation mediated by Topoisomerase II is required to separate the interlinked sister chromatids post-replication. SGS1, a yeast homolog of the human RecQ family of helicases interacts with Topoisomerase II and plays a role in chromosome segregation, but this functional interaction has yet to be identified in higher organisms. Here, we report a physical and functional interaction of Topoisomerase IIα with RECQL5, one of five mammalian RecQ helicases, during DNA replication. Direct interaction of RECQL5 with Topoisomerase IIα stimulates the decatenation activity of Topoisomerase IIα. Consistent with these observations, RECQL5 co-localizes with Topoisomerase IIα during S-phase of the cell cycle. Moreover, cells with stable depletions of RECQL5 display a slow proliferation rate, a G2/M cell cycle arrest and late S-phase cycling defects. Metaphase spreads generated from RECQL5-depleted cells exhibit undercondensed and entangled chromosomes. Further, RECQL5-depleted cells activate a G2/M checkpoint and undergo apoptosis. These phenotypes are similar to those observed when Topoisomerase II catalytic activity is inhibited. These results reveal an important role for RECQL5 in the maintenance of genomic stability and a new insight into the decatenation process.

Newly identified CHO ERCC3/XPB mutations and phenotype characterization.

Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron-exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5' incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages approximately 8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER.

Characterization of ERCC3 mutations in the Chinese hamster ovary 27-1, UV24 and MMC-2 cell lines.

Mutation of the XPB gene in humans gives rise to the distinct, autosomal recessive disorder, with a striking clinical heterogeneity: xeroderma pigmentosum associated with Cockayne's syndrome and trichothiodystrophy. XPB is a subunit of a multifunctional RNA polymerase II general initiation factor TFIIH and codes for 3'-->5' DNA helicase essential for both nucleotide excision repair (NER) and transcription. Since XPB defective human disease is extremely rare, Chinese hamster ovary (CHO) mutant cell lines belonging to the 3rd rodent complementation group (the hamster ERCC3 gene is the homologue of the human XPB gene) are a unique resource for analyzing structure-function relationships in the ERCC3/XPB protein. We have amplified, cloned and sequenced the ERCC3 genes from wild type and 27-1, UV24 and MMC-2 CHO mutant cell lines and identified the sites of the respective mutations. 27-1 mutant has an A1075G transition (K359E) located at the very beginning of the Ia helicase domain which causes deficiency in open complex formation and in 3', 5' and dual incisions during NER. UV24 cell line has two mutations. First, it is a T1144C transition (S382P) located behind the Ia helicase domain in a region responsible for ERCC3 binding to XPG, p62 and p44. Second mutation is identical with a mutation in MMC-2 mutant. It is a C2215T transition (Q739STOP) causing the truncation of the C-terminus of the protein, responsible for the 5' incision, by 44 amino acids. All mutant cell lines are unable to recover RNA synthesis after 10Jm(-2) UV, suggesting a defect in transcription-coupled repair. Their limited global NER capacity measured by a single-cell gel electrophoresis assay (0.25Jm(-2)) varies from 6% to 11%.

Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

BACKGROUND: The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA). CONCLUSIONS/SIGNIFICANCE: These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

Human RECQL5 participates in the removal of endogenous DNA damage.

Human RECQL5 is a member of the RecQ helicase family, which maintains genome stability via participation in many DNA metabolic processes, including DNA repair. Human cells lacking RECQL5 display chromosomal instability. We find that cells depleted of RECQL5 are sensitive to oxidative stress, accumulate endogenous DNA damage, and increase the cellular poly(ADP-ribosyl)ate response. In contrast to the RECQ helicase family members WRN, BLM, and RECQL4, RECQL5 accumulates at laser-induced single-strand breaks in normal human cells. RECQL5 depletion affects the levels of PARP-1 and XRCC1, and our collective results suggest that RECQL5 modulates and/or directly participates in base excision repair of endogenous DNA damage, thereby promoting chromosome stability in normal human cells.

Cockayne syndrome group B protein stimulates repair of formamidopyrimidines by NEIL1 DNA glycosylase.

Cockayne syndrome (CS) is a premature aging condition characterized by sensitivity to UV radiation. However, this phenotype does not explain the progressive neurodegeneration in CS patients. It could be due to the hypersensitivity of CSB-deficient cells to oxidative stress. So far most studies on the role of CSB in repair of oxidatively induced DNA lesions have focused on 7,8-dihydro-8-oxoguanine. This study examines the role of CSB in the repair of formamidopyrimidines 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde), which are substrates for endonuclease VIII-like (NEIL1) DNA glycosylase. Results presented here show that csb(-/-) mice have a higher level of endogenous FapyAde and FapyGua in DNA from brain and kidney than wild type mice as well as higher levels of endogenous FapyAde in genomic DNA and mtDNA from liver. In addition, CSB stimulates NEIL1 incision activity in vitro, and CSB and NEIL1 co-immunoprecipitate and co-localize in HeLa cells. When CSB and NEIL1 are depleted from HeLa cells by short hairpin RNA knockdown, repair of induced FapyGua is strongly inhibited. These results suggest that CSB plays a role in repair of formamidopyrimidines, possibly by interacting with and stimulating NEIL1, and that accumulation of such modifications may have a causal role in the pathogenesis of CS.