Involvement of protein kinase A in the phosphorylation of spermatidal protein TP2 and its effect on DNA condensation.

Rat spermatidal protein TP2 is rich in serine residues and has several potential sites for phosphorylation by different protein kinases. Recombinant TP2 is phosphorylated upon incubation in vitro with salt extract of testicular sonication resistant nuclei (SRN) (representing elongating and elongated spermatids). The major phosphorylation sites were localized to the C-terminal, V8 protease-derived, fragment (residues 87-114). Phosphorylation experiments with the wild type and different site-specific mutants of TP2 revealed that serine 109 and threonine 101 are the phosphorylation sites. Phosphorylation of the C-terminal fragment of TP2 was also demonstrated in vivo. Phosphorylation was not stimulated by either protein kinase C activators or cGMP but was inhibited by protein kinase A inhibitor (PKI) peptide, showing the involvement of protein kinase A in the phosphorylation of TP2. Phosphorylation of TP2 greatly reduced its DNA condensation property. TP2 when complexed with DNA was not a good substrate for phosphorylation by PKA. Dephosphorylation of the DNA-TP2 complex by calf intestinal alkaline phosphatase restored the DNA condensation property to a level equivalent to that observed with TP2. The physiological significance of the phosphorylation-dephosphorylation cycle is discussed with reference to the two-domain model of TP2.     

Identification of two novel zinc finger modules and nuclear localization signal in rat spermatidal protein TP2 by site-directed mutagenesis

Spermatidal protein TP2, which appears transiently during stages 12-16 of mammalian spermiogenesis, is a DNA condensing zinc metalloprotein with a preference to GC-rich DNA. We have carried out a detailed site-directed mutagenesis analysis of rat spermatidal protein TP2 to delineate the amino acid residues involved in coordination with two atoms of zinc. Two zinc fingers modules have been identified involving 4 histidine and 4 cysteine residues, respectively. The modular structure of the two zinc fingers identified in TP2 define a new class of zinc finger proteins that do not fall into any of the known classes of zinc fingers. Transfection experiments with COS-7 cells using wild type and the two zinc finger pocket mutants have shown that TP2 preferentially localizes to nucleolus. The nuclear localization signal in TP2 was identified to be (87)GKVSKRKAV(95) present in the C-terminal third of TP2 as a part of an extended NoLS sequence.     

Stimulation of the UvrABC enzyme-catalyzed repair reactions by the UvrD protein (DNA helicase II).

An in vitro assay system was constructed using highly purified preparations of UvrA, UvrB, UvrC, UvrD proteins and DNA polymerase I, the objective being to analyse the role of UvrD protein in excision repair of UV-induced DNA damage. UvrABC enzyme-initiated repair synthesis was greatly enhanced by the addition of UvrD protein to the reaction mixture. Further analysis revealed that UvrD protein stimulated introduction of strand breaks in irradiated DNA by UvrABC enzyme but had no effect on the DNA polymerase I reaction. Thus, the site of action of UvrD protein is probably at the incision-excision step and not in later steps in excision repair.     

Specificity of protein interactions mediated by BRCT domains of the XRCC1 DNA repair protein

Protein interactions critical to DNA repair and cell cycle control systems are often coordinated by modules that belong to a superfamily of structurally conserved BRCT domains. Because the mechanisms of BRCT interactions and their significance are not well understood, we sought to define the affinity and specificity of those BRCT modules that orchestrate base excision repair and single-strand break repair. Common to these pathways is the essential XRCC1 DNA repair protein, which interacts with at least nine other proteins and DNA. Here, we characterized the interactions of four purified BRCT domains, two from XRCC1 and their two partners from DNA ligase IIIalpha and poly(ADP-ribosyl) polymerase 1. A monoclonal antibody was selected that recognizes the ligase IIIalpha BRCT domain, but not the other BRCT domains, and was used to capture the relevant ligase IIIalpha BRCT complex. To examine the assembly states of isolated BRCT domains and pairwise domain complexes, we used size-exclusion chromatography coupled with on-line light scattering. This analysis indicated that isolated BRCT domains form homo-oligomers and that the BRCT complex between the C-terminal XRCC1 domain and the ligase IIIalpha domain is a heterotetramer with 2:2 stoichiometry. Using affinity capture and surface plasmon resonance methods, we determined that specific heteromeric interactions with high nanomolar dissociation constants occur between pairs of cognate BRCT domains. A structural model for a XRCC1 x DNA ligase IIIalpha heterotetramer is proposed as a core base excision repair complex, which constitutes a scaffold for higher order complexes to which other repair proteins and DNA are brought into proximity.     

Modulation of error-prone double-strand break repair in mammalian chromosomes by DNA mismatch repair protein Mlh1

We assayed error-prone double-strand break (DSB) repair in wild-type and isogenic Mlh1-null mouse embryonic fibroblasts containing a stably integrated DSB repair substrate. The substrate contained a thymidine kinase (tk) gene fused to a neomycin-resistance (neo) gene; the tk-neo fusion gene was disrupted in the tk portion by a 22bp oligonucleotide containing the 18 bp recognition site for endonuclease I-SceI. Following DSB-induction by transient expression of I-SceI endonuclease, cells that repaired the DSB by error-prone nonhomologous end-joining (NHEJ) and restored the correct reading frame to the tk-neo fusion gene were recovered by selecting for G418-resistant clones. The number of G418-resistant clones induced by I-SceI expression did not differ significantly between wild-type and Mlh1-deficient cells. While most DSB repair events were consistent with simple NHEJ in both wild-type and Mlh1-deficient cells, complex repair events were more common in wild-type cells. Furthermore, genomic deletions associated with NHEJ events were strikingly larger in wild-type versus Mlh1-deficient cells. Additional experiments revealed that the stable transfection efficiency of Mlh1-null cells is higher than that of wild-type cells. Collectively, our results suggest that Mlh1 modulates error-prone NHEJ by inhibiting the annealing of DNA ends containing noncomplementary base pairs or by promoting the annealing of microhomologies.     

Stimulation with quaterin of DNA replication and repair

Quaterine [3-(2,2,3-trimethylhydrasinium)propionate] possessing a wide spectrum of physiological activity has been studied for its effect on the intensity of replicative and reparative DNA synthesis in different rat tissues (liver, thymus, heart, intestine mucosa and spleen) in order to investigate molecular mechanisms of its action. It is shown that the pronounced stimulation of DNA synthesis in all tissues, as a rule, takes place 1-6h after quaterine administration in doses of 25 and 100 mg/kg. The estimation of the given compound effect on DNA synthesis after its multiple administration to animals (for 5, 10, 15 days in a dose of 100 mg/kg) permits supposing that 3-(2,2,2-trimethylhydrasinium)propionate is able of providing either stable proliferation of cells (thymus, spleen) or their hyperplasia and polyploidization (heart, liver). The data obtained make it possible to explain (to some extent) quaterine ability to activate immune responses, to stimulate healing of wounds and burns.     

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.     

DNA binding and nucleotide flipping by the human DNA repair protein AGT

O(6)-alkylguanine-DNA alkyltransferase (AGT), or O(6)-methylguanine-DNA methyltransferase (MGMT), prevents mutations and apoptosis resulting from alkylation damage to guanines. AGT irreversibly transfers the alkyl lesion to an active site cysteine in a stoichiometric, direct damage reversal pathway. AGT expression therefore elicits tumor resistance to alkylating chemotherapies, and AGT inhibitors are in clinical trials. We report here structures of human AGT in complex with double-stranded DNA containing the biological substrate O(6)-methylguanine or crosslinked to the mechanistic inhibitor N(1),O(6)-ethanoxanthosine. The prototypical DNA major groove-binding helix-turn-helix (HTH) motif mediates unprecedented minor groove DNA binding. This binding architecture has advantages for DNA repair and nucleotide flipping, and provides a paradigm for HTH interactions in sequence-independent DNA-binding proteins like RecQ and BRCA2. Structural and biochemical results further support an unpredicted role for Tyr114 in nucleotide flipping through phosphate rotation and an efficient kinetic mechanism for locating alkylated bases.     

Stimulation of flap endonuclease-1 by the Bloom's syndrome protein

Bloom's syndrome (BS) is a rare autosomal recessive genetic disorder associated with genomic instability and an elevated risk of cancer. Cellular features of BS include an accumulation of abnormal replication intermediates and increased sister chromatid exchange. Although it has been suggested that the underlying defect responsible for hyper-recombination in BS cells is a temporal delay in the maturation of DNA replication intermediates, the precise role of the BS gene product, BLM, in DNA metabolism remains elusive. We report here a novel interaction of the BLM protein with the human 5'-flap endonuclease/5'-3' exonuclease (FEN-1), a genome stability factor involved in Okazaki fragment processing and DNA repair. BLM protein stimulates both the endonucleolytic and exonucleolytic cleavage activity of FEN-1 and this functional interaction is independent of BLM catalytic activity. BLM and FEN-1 are associated with each other in human nuclei as shown by their reciprocal co-immunoprecipitation from HeLa nuclear extracts. The BLM-FEN-1 physical interaction is mediated through a region of the BLM C-terminal domain that shares homology with the FEN-1 interaction domain of the Werner syndrome protein, a RecQ helicase family member homologous to BLM. This study provides the first evidence for a direct interaction of BLM with a human nucleolytic enzyme. We suggest that functional interactions between RecQ helicases and Rad2 family nucleases serve to process DNA substrates that are intermediates in DNA replication and repair.     

Suppression of UV-induced apoptosis by the human DNA repair protein XPG

The severe xeroderma pigmentosum/Cockayne syndrome (XP/CS) syndrome is caused by mutations in the XPB, XPD and XPG genes that encode the helicase subunits of TFIIH and the 3' endonuclease of nucleotide excision repair (NER). Because XPB and XPD have been implicated in p53-mediated apoptosis, we examined the possible involvement of XPG in this process. After ultraviolet light (UV) irradiation, primary fibroblasts of XP complementation group G (XP-G) individuals with CS enter apoptosis more readily than other NER-deficient cells, but this is unlinked to unrepaired damage. These XP-G/CS cells accumulate p53 post-UV but they fail to accumulate the 90/92 kDa isoforms of Mdm2 and their cellular distribution of Mdm2 is impaired. Apoptosis levels revert to wild type, Mdm2 90/92 kDa isoforms accumulate, and Mdm2 regains its normal post-UV nuclear location in transduced XP-G/CS cells expressing wild-type XPG, but not an XPG catalytic site mutant. These results suggest that XPG suppresses UV-induced apoptosis and that this suppression, most simply, requires its endonuclease function.     

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.     

Interaction of HIV-1 integrase with DNA repair protein hRad18

We have previously shown that human immunodeficiency virus-1 (HIV-1) integrase is an unstable protein and a substrate for the N-end rule degradation pathway. This degradation pathway shares its ubiquitin-conjugating enzyme, Rad6, with the post-replication/translesion DNA repair pathway. Because DNA repair is thought to play an essential role in HIV-1 integration, we investigated whether other molecules of this DNA repair pathway could interact with integrase. We observed that co-expression of human Rad18 induced the accumulation of an otherwise unstable form of HIV-1 integrase. This accumulation occurred even though hRAD18 possesses a RING finger domain, a structure that is generally associated with E3 ubiquitin ligase function and protein degradation. Evidence for an interaction between integrase and hRad18 was obtained through reciprocal co-immunoprecipitation. Moreover we found that a 162-residue region of hRad18 (amino acids 65-226) was sufficient for both integrase stabilization and interaction. Finally, we observed that HIV-1 integrase co-localized with hRad18 in nuclear structures in a subpopulation of co-transfected cells. Taken together, these findings identify hRad18 as a novel interacting partner of HIV-1 integrase and suggest a role for post-replication/translesion DNA repair in the retroviral integration process.     

TP53 is not required for the constitutive or induced repair of DNA damage produced by ionizing radiation at the G1/S-phase border

We have previously described a novel DNA repair response that is induced in cells irradiated with ionizing radiation at the G1/S-phase border and is characterized by the formation of very long repair patches (VLRP) containing at least 150 nucleotides. In the current study, we examined whether there is a requirement for TP53 in this induced repair process. We find that in normal cells, the endogenous levels of TP53 are elevated at the G1/S-phase border, and that these levels are not further increased after irradiation with 5 Gy. In cells expressing the E6 oncoprotein of human papillomavirus, which inactivates TP53 function, there is a greatly accentuated induction of the VLRP that nearly masks the constitutive repair response. Incubation of cells in the presence of cycloheximide, which inhibits the induced repair, reveals the presence of the constitutive repair patches. All cells examined continue to replicate their DNA after exposure to ionizing radiation. In contrast, cells irradiated with UV radiation at the G1/S-phase border show an induction of TP53 protein and halt DNA synthesis, but do not induce the VLRP. Our results show that TP53 is not required for the constitutive or induced repair of DNA damage induced by ionizing radiation. In addition, these results suggest that TP53 may suppress the formation of VLRP and that the progression of cells through S phase after exposure to ionizing radiation signals the induced repair response.