人类polⅡ启动子的识别

依据基因启动子区和非启动子区碱基分布的特征,应用基于多样性增量的二次判别分析 (IDQD),对人类polⅡ启动子进行识别,识别精度达到90％以上的水平,优于其他已发表的 (包括SVM分类器等) 识别算法. 使用IDQD算法也能对转录起始位点 (TSS) 进行较准确的预测,10-fold交叉检验结果的敏感性和特异性分别为86％和91％. 这些结果表明IDQD是一个有效的分类器.     

Translesion DNA synthesis catalyzed by human pol eta and pol kappa across 1,N6-ethenodeoxyadenosine

1,N(6)-Ethenodeoxyadenosine, a DNA adduct generated by exogenous and endogenous sources, severely blocks DNA synthesis and induces miscoding events in human cells. To probe the mechanism for in vivo translesion DNA synthesis across this adduct, in vitro primer extension studies were conducted using newly identified human DNA polymerases (pol) eta and kappa, which have been shown to catalyze translesion DNA synthesis past several DNA lesions. Steady-state kinetic analyses and analysis of translesion products have revealed that the synthesis is >100-fold more efficient with pol eta than with pol kappa and that both error-free and error-prone syntheses are observed with these enzymes. The miscoding events include both base substitution and frameshift mutations. These results suggest that both polymerases, particularly pol eta, may contribute to the translesion DNA synthesis events observed for 1,N(6)-ethenodeoxyadenosine in human cells.     

When pol I goes into high gear: processive DNA synthesis by pol I in the cell

Pol I is the most abundant polymerase in E. coli and plays an important role in short patch repair. In accord with this role in the cell, the purified polymerase exhibits low processivity and high fidelity in vitro. Pol I is also the polymerase responsible for leader strand synthesis during ColE1 plasmid replication. In a previous publication, we described the generation of a highly error-prone DNA polymerase I. Expression of this mutant Pol I results in errors during the replication of a ColE1 plasmid. The distribution and spectrum of mutations in the ColE1 plasmid sequence downstream the ori indicates that Pol I is capable of more processive replication in vivo than previously accepted. Here, we review evidence suggesting that Pol I may be recruited into a replisome-like holoenzyme and speculate that processive DNA replication by Pol I may play a role in recombination-dependent DNA replication in the cell.     

DNA binding properties of human pol gammaB

We have recently reported the crystal structure of the accessory subunit of mitochondrial DNA polymerase, pol gammaB, and identified a region of the protein involved in DNA binding. The DNA employed in previous studies was presumed to be single-stranded, because it was generated by single-sided PCR. Further characterization of this DNA indicated that, due to a strand transfer event during synthesis by single-sided PCR, the DNA adopts a double-stranded hairpin conformation under native conditions. We used a series of double- and single-stranded oligonucleotides of different lengths to confirm that human pol gammaB prefers to bind double-stranded DNA longer than 40 bp with little apparent sequence specificity. Site-specific deletion mutagenesis identified clusters of basic residues in two surface loops required for DNA binding located on opposite sides of the symmetrical pol gammaB dimer. A heterodimer of pol gammaB that contains one mutant and one wild-type DNA binding region was shown to be unable to bind double-stranded DNA, suggesting that a single DNA molecule must contact both DNA binding sites in the pol gammaB dimer. The ability to bind double-stranded DNA is not essential for pol gammaB stimulation of pol gammaA activity in vitro, but may play a role in DNA replication or repair.     

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon ) complex. Demonstration of a dimeric pol epsilon

Saccharomyces cerevisiae DNA polymerase epsilon (pol epsilon) is essential for chromosomal replication. A major form of pol epsilon purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p.Dpb2p and Dpb3p.Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol epsilon may be dimeric in vivo. Gel filtration of the Pol2p.Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p.Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.     

Association of DNA polymerase mu (pol mu) with Ku and ligase IV: role for pol mu in end-joining double-strand break repair

Mammalian DNA polymerase mu (pol mu) is related to terminal deoxynucleotidyl transferase, but its biological role is not yet clear. We show here that after exposure of cells to ionizing radiation (IR), levels of pol mu protein increase. pol mu also forms discrete nuclear foci after IR, and these foci are largely coincident with IR-induced foci of gammaH2AX, a previously characterized marker of sites of DNA double-strand breaks. pol mu is thus part of the cellular response to DNA double-strand breaks. pol mu also associates in cell extracts with the nonhomologous end-joining repair factor Ku and requires both Ku and another end-joining factor, XRCC4-ligase IV, to form a stable complex on DNA in vitro. pol mu in turn facilitates both stable recruitment of XRCC4-ligase IV to Ku-bound DNA and ligase IV-dependent end joining. In contrast, the related mammalian DNA polymerase beta does not form a complex with Ku and XRCC4-ligase IV and is less effective than pol mu in facilitating joining mediated by these factors. Our data thus support an important role for pol mu in the end-joining pathway for repair of double-strand breaks.     

HIV-1 Clade B pol Evolution following Primary Infection

Objective

Characterize intra-individual HIV-1 subtype B pol evolution in antiretroviral naive individuals.

Design

Longitudinal cohort study of individuals enrolled during primary infection.

Methods

Eligible individuals were antiretroviral naïve participants enrolled in the cohort from December 1997-December 2005 and having at least two blood samples available with the first one collected within a year of their estimated date of infection. Population-based pol sequences were generated from collected blood samples and analyzed for genetic divergence over time in respect to dual infection status, HLA, CD4 count and viral load.

Results

93 participants were observed for a median of 1.8 years (Mean = 2.2 years, SD = 1.9 years). All participants classified as mono-infected had less than 0.7% divergence between any two of their pol sequences using the Tamura-Nei model (TN93), while individuals with dual infection had up to 7.0% divergence. The global substitution rates (substitutions/nucleotide/year) for mono and dually infected individuals were significantly different (p<0.001); however, substitution rates were not associated with HLA haplotype, CD4 or viral load.

Conclusions

Even after a maximum of almost 9 years of follow-up, all mono-infected participants had less than 1% divergence between baseline and longitudinal sequences, while participants with dual infection had 10 times greater divergence. These data support the use of HIV-1 pol sequence data to evaluate transmission events, networks and HIV-1 dual infection.