62 research outputs found
Interaction of hHR23 with S5a. The ubiquitin-like domain of hHR23 mediates interaction with S5a subunit of 26 S proteasome
hHR23B is one of two human homologs of the Saccharomyces cerevisiae
nucleotide excision repair (NER) gene product RAD23 and a component of a
protein complex that specifically complements the NER defect of xeroderma
pigmentosum group C (XP-C) cell extracts in vitro. Although a small
proportion of hHR23B is tightly complexed with the XP-C responsible gene
product, XPC protein, a vast majority exists as an XPC-free form,
indicating that hHR23B has additional functions other than NER in vivo.
Here we demonstrate that the human NER factor hHR23B as well as another
human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit
of the human 26 S proteasome using the yeast two-hybrid system.
Furthermore, hHR23 proteins were detected with S5a at the position where
26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100
extracts. Intriguingly, hHR23B showed the inhibitory effect on the
degradation of (125)I-lysozyme in the rabbit reticulocyte lysate. hHR23
proteins thus appear to associate with 26 S proteasome in vivo. From
co-precipitation experiments using several series of deletion mutants, we
defined the domains in hHR23B and S5a that mediate this interaction. From
these results, we propose that part of hHR23 proteins are involved in the
proteolytic pathway in cells
129-derived Strains of Mice Are Deficient in DNA Polymerase Ī¹ and Have Normal Immunoglobulin Hypermutation
Recent studies suggest that DNA polymerase Ī· (polĪ·) and DNA polymerase Ī¹ (polĪ¹) are involved in somatic hypermutation of immunoglobulin variable genes. To test the role of polĪ¹ in generating mutations in an animal model, we first characterized the biochemical properties of murine polĪ¹. Like its human counterpart, murine polĪ¹ is extremely error-prone when catalyzing synthesis on a variety of DNA templates in vitro. Interestingly, when filling in a 1 base-pair gap, DNA synthesis and subsequent strand displacement was greatest in the presence of both pols Ī¹ and Ī·. Genomic sequence analysis of Poli led to the serendipitous discovery that 129-derived strains of mice have a nonsense codon mutation in exon 2 that abrogates production of polĪ¹. Analysis of hypermutation in variable genes from 129/SvJ (Poliā/ā) and C57BL/6J (Poli+/+) mice revealed that the overall frequency and spectrum of mutation were normal in polĪ¹-deficient mice. Thus, either polĪ¹ does not participate in hypermutation, or its role is nonessential and can be readily assumed by another low-fidelity polymerase
Characterization of a Y-Family DNA Polymerase eta from the Eukaryotic Thermophile Alvinella pompejana
Human DNA polymerase Ī· (HsPolĪ·) plays an important role in translesion synthesis (TLS), which allows for replication past DNA damage such as UV-induced cis-syn cyclobutane pyrimidine dimers (CPDs). Here, we characterized ApPolĪ· from the thermophilic worm Alvinella pompejana, which inhabits deep-sea hydrothermal vent chimneys. ApPolĪ· shares sequence homology with HsPolĪ· and contains domains for binding ubiquitin and proliferating cell nuclear antigen. Sun-induced UV does not penetrate Alvinella's environment; however, this novel DNA polymerase catalyzed efficient and accurate TLS past CPD, as well as 7,8-dihydro-8-oxoguanine and isomers of thymine glycol induced by reactive oxygen species. In addition, we found that ApPolĪ· is more thermostable than HsPolĪ·, as expected from its habitat temperature. Moreover, the activity of this enzyme was retained in the presence of a higher concentration of organic solvents. Therefore, ApPolĪ· provides a robust, human-like PolĪ· that is more active after exposure to high temperatures and organic solvents
Photosensitized [2 + 2] cycloaddition of N-acetylated cytosine affords stereoselective formation of cyclobutane pyrimidine dimer
Photocycloaddition between two adjacent bases in DNA produces a cyclobutane pyrimidine dimer (CPD), which is one of the major UV-induced DNA lesions, with either the cis-syn or trans-syn structure. In this study, we investigated the photosensitized intramolecular cycloaddition of partially-protected thymidylyl-(3ā²ā5ā²)-N4-acetyl-2ā²-deoxy-5-methylcytidine, to clarify the effect of the base modification on the cycloaddition reaction. The reaction resulted in the stereoselective formation of the trans-syn CPD, followed by hydrolysis of the acetylamino group. The same result was obtained for the photocycloaddition of thymidylyl-(3ā²ā5ā²)-N4-acetyl-2ā²-deoxycytidine, whereas both the cis-syn and trans-syn CPDs were formed from thymidylyl-(3ā²ā5ā²)-thymidine. Kinetic analyses revealed that the activation energy of the acid-catalyzed hydrolysis is comparable to that reported for the thymine-cytosine CPD. These findings provided a new strategy for the synthesis of oligonucleotides containing the trans-syn CPD. Using the synthesized oligonucleotide, translesion synthesis by human DNA polymerase Ī· was analyzed
Structure and mechanism of human DNA polymerase Ī·
The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites
Simultaneous disruption of two DNA polymerases, PolĪ· and PolĪ¶, in Avian DT40 cells unmasks the role of PolĪ· in cellular response to various DNA lesions
Replicative DNA polymerases are frequently stalled by DNA lesions. The resulting replication blockage is released by homologous recombination (HR) and translesion DNA synthesis (TLS). TLS employs specialized TLS polymerases to bypass DNA lesions. We provide striking in vivo evidence of the cooperation between DNA polymerase Ī·, which is mutated in the variant form of the cancer predisposition disorder xeroderma pigmentosum (XP-V), and DNA polymerase Ī¶ by generating POLĪ·ā/ā/POLĪ¶ā/ā cells from the chicken DT40 cell line. POLĪ¶ā/ā cells are hypersensitive to a very wide range of DNA damaging agents, whereas XP-V cells exhibit moderate sensitivity to ultraviolet light (UV) only in the presence of caffeine treatment and exhibit no significant sensitivity to any other damaging agents. It is therefore widely believed that PolĪ· plays a very specific role in cellular tolerance to UV-induced DNA damage. The evidence we present challenges this assumption. The phenotypic analysis of POLĪ·ā/ā/POLĪ¶ā/ā cells shows that, unexpectedly, the loss of PolĪ· significantly rescued all mutant phenotypes of POLĪ¶ā/ā cells and results in the restoration of the DNA damage tolerance by a backup pathway including HR. Taken together, PolĪ· contributes to a much wide range of TLS events than had been predicted by the phenotype of XP-V cells
Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication
The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, GĀ·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions
Critical amino acids in human DNA polymerases Ī· and Īŗ involved in erroneous incorporation of oxidized nucleotides
Oxidized DNA precursors can cause mutagenesis and carcinogenesis when they are incorporated into the genome. Some human Y-family DNA polymerases (Pols) can effectively incorporate 8-oxo-dGTP, an oxidized form of dGTP, into a position opposite a template dA. This inappropriate G:A pairing may lead to transversions of A to C. To gain insight into the mechanisms underlying erroneous nucleotide incorporation, we changed amino acids in human PolĪ· and PolĪŗ proteins that might modulate their specificity for incorporating 8-oxo-dGTP into DNA. We found that Arg61 in PolĪ· was crucial for erroneous nucleotide incorporation. When Arg61 was substituted with lysine (R61K), the ratio of pairing of dA to 8-oxo-dGTP compared to pairing of dC was reduced from 660:1 (wild-type PolĪ·) to 7 : 1 (R61K). Similarly, Tyr112 in PolĪŗ was crucial for erroneous nucleotide incorporation. When Tyr112 was substituted with alanine (Y112A), the ratio of pairing was reduced from 11: 1 (wild-type PolĪŗ) to almost 1: 1 (Y112A). Interestingly, substitution at the corresponding position in PolĪ·, i.e. Phe18 to alanine, did not alter the specificity. These results suggested that amino acids at distinct positions in the active sites of PolĪ· and PolĪŗ might enhance 8-oxo-dGTP to favor the syn conformation, and thus direct its misincorporation into DNA
Mechanisms of accurate translesion synthesis by human DNA polymerase Ī·
The XPV (xeroderma pigmentosum variant) gene encodes human DNA polymerase Ī· (pol Ī·), which is involved in the replication of damaged DNA. Pol Ī· catalyzes efficient and accurate translesion synthesis past cis-syn cyclobutane di-thymine lesions. Here we show that human pol Ī· 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 Ī· 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 Ī· could not continue chain elongation. In contrast, after incorporating the correct nucleotide opposite a lesion, pol Ī· could continue chain elongation, whereas pol Ī± could not. Thus, the fidelity of translesion synthesis by human pol Ī· 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
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