Biomedical subjects

Igor V Shevelev

Publications and source records attributed to Igor V Shevelev.

6 recordsLinked to original sources

PubMed2004

De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase.

DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3'OH end of the primer along the template. On the other hand terminal deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3'OH end of a DNA strand, without the need of a template. Pol lambda and pol micro are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol micro and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol micro and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.

Base Sequence↗

PubMed2004

Involvement of an X family DNA polymerase in double-stranded break repair in the radioresistant organism Deinococcus radiodurans.

DNA polymerases of the X family have been implicated in a variety of DNA repair processes in eukaryotes. Here we show that Deinococcus radiodurans, a highly radioresistant bacterium able to mend hundreds of radiation-induced double-stranded DNA breaks, expresses a DNA polymerase belonging to the X family. This novel bacterial polymerase, named PolX(Dr), was identified as the product of the Deinococcal DR0467 gene. The purified PolX(Dr) protein possesses a DNA polymerase activity that is stimulated by MnCl2, a property of the X family DNA polymerases. Antibodies raised against PolX(Dr) recognized human pol lambda, rat pol beta and yeast Pol4 and, conversely, antibodies raised against these proteins recognized PolX(Dr). This immunological cross-reactivity suggests a high degree of structural conservation among the polymerases of the X family. Lack of PolX(Dr) reduced the rate of repair of double-stranded DNA breaks and increased cell sensitivity to gamma-rays. PolX(Dr) thus appears to play an important role in double-stranded DNA break repair in D. radiodurans.

Animals↗

PubMed2003

Human DNA polymerase lambda possesses terminal deoxyribonucleotidyl transferase activity and can elongate RNA primers: implications for novel functions.

DNA polymerase lambda is a novel enzyme of the family X of DNA polymerases. The recent demonstration of an intrinsic 5'-deoxyribose-5'-phosphate lyase activity, a template/primer dependent polymerase activity, a distributive manner of DNA synthesis and sequence similarity to DNA polymerase beta suggested a novel beta-like enzyme. All these properties support a role of DNA polymerase lambda in base excision repair. On the other hand, the biochemical properties of the polymerisation activity of DNA polymerase lambda are still largely unknown. Here we give evidence that human DNA polymerase lambda has an intrinsic terminal deoxyribonucleotidyl transferase activity that preferentially adds pyrimidines onto 3'OH ends of DNA oligonucleotides. Furthermore, human DNA polymerase lambda efficiently elongates an RNA primer hybridized to a DNA template. These two novel properties of human DNA polymerase lambda might suggest additional roles for this enzyme in DNA replication and repair processes.

Base Sequence↗

PubMed2002

DNA polymerase lambda from calf thymus preferentially replicates damaged DNA.

A new gene (POLL), has been identified encoding the novel DNA polymerase lambda and mapped to mouse chromosome 19 and at human chromosome 10. DNA polymerase lambda contains all the critical residues involved in DNA binding, nucleotide binding, nucleotide selection, and catalysis of DNA polymerization and has been assigned to family X based on sequence homology with polymerase beta, lambda, mu, and terminal deoxynucleotidyltransferase. Here we describe a purification of DNA polymerase lambda from calf thymus that preferentially can replicate damaged DNA. By testing polymerase activity on non-damaged and damaged DNA, DNA polymerase lambda was purified trough five chromatographic steps to near homogeneity and identified as a 67-kDa polypeptide that cross-reacted with monoclonal antibodies against DNA polymerase beta and polyclonal antibodies against DNA polymerase lambda. DNA polymerase lambda had no detectable nuclease activities and, in contrast to DNA polymerase beta, was aphidicolin-sensitive. DNA polymerase lambda was a 6-fold more accurate enzyme in an M13mp2 forward mutation assay and 5-fold more accurate in an M13mp2T90 reversion system than human recombinant DNA polymerase beta. The biochemical properties of the calf thymus DNA polymerase lambda, described here for the first time, are discussed in relationship to the proposed role for this DNA polymerase in vivo.

Animals↗

PubMed2002

The TREX2 3'-->5' exonuclease physically interacts with DNA polymerase delta and increases its accuracy.

Proofreading function by the 3'-->5' exonuclease of DNA polymerase delta (pol delta) is consistent with the observation that delta efficiency of the associated exonuclease can lead to a strong mutation phenotype, high error rates during DNA replication, and ultimately cancer. We have isolated pol delta from isotonic (pol delta(i)) and detergent (pol delta(d)) calf thymus extracts. Pol delta had a 20-fold higher ratio of exonuclease to DNA polymerase than pol deltai. This was due to the physical association of the TREX2 3'-->5' exonuclease to pol delta(d), which was missing from pol delta(i). Pol delta(d) was fivefold more accurate than pol i under error-prone conditions (1 mM dGTP and 20 dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate than pol delta(i) under error-prone conditions (1 mM dGTP and 20 microM dATP, dCTP, and dTTP) in a M13mp2 DNA forward mutation assay, and fourfold more accurate in an M13mp2T90 reversion assay. Under error-free conditions (20 microM each of the four dNTPs), however, both polymerases showed equal fidelity. Our data suggested that autonomous 3' --> 5' exonucleases, such as TREX2, through its association with pol delta can guarantee high fidelity under difficult conditions in the cell (e.g., imbalance of dNTPs) and can add to the accuracy of the DNA replication machinery, thus preventing mutagenesis.

Animals↗

PubMed2002

The 3' 5' exonucleases.

Over the past few years, several new 3' 5' exonucleases have been identified. In vitro studies of these enzymes have uncovered much about their potential functions in vivo, and certain organisms with a defect in 3' 5' exonucleases have an increased susceptibility to cancer, especially under conditions of stress. Here, we look at not only the newly discovered enzymes, but also at the roles of other 3' 5' exonucleases in the quality control of DNA synthesis, where they act as proofreading exonucleases for DNA polymerases during DNA replication, repair and recombination.

Amino Acid Sequence↗