Biomedical subjects

R D Kolodner

Publications and source records attributed to R D Kolodner.

97 records · Page 6Linked to original sources

PubMed1988

Analysis of the recE locus of Escherichia coli K-12 by use of polyclonal antibodies to exonuclease VIII.

Exonuclease VIII (exoVIII) of Escherichia coli has been purified from a strain carrying a plasmid-encoded recE gene by using a new procedure. This procedure yielded 30 times more protein per gram of cells, and the protein had a twofold higher specific activity than the enzyme purified by the previously published procedure (J. W. Joseph and R. Kolodner, J. Biol. Chem. 258:10411-10417, 1983). The sequence of the 12 N-terminal amino acids was also obtained and found to correspond to one of the open reading frames predicted from the nucleic acid sequence of the recE region of Rac (C. Chu, A. Templin, and A. J. Clark, manuscript in preparation). Polyclonal antibodies directed against purified exoVIII were also prepared. Cell-free extracts prepared from strains containing a wide range of chromosomal- or plasmid-encoded point, insertion, and deletion mutations which result in expression of exoVIII were examined by Western blot (immunoblot) analysis. This analysis showed that two point sbcA mutations (sbcA5 and sbcA23) and the sbc insertion mutations led to the synthesis of the 140-kilodalton (kDa) polypeptide of wild-type exoVIII. Plasmid-encoded partial deletion mutations of recE reduced the size of the cross-reacting protein(s) in direct proportion to the size of the deletion, even though exonuclease activity was still present. The analysis suggests that 39 kDa of the 140-kDa exoVIII subunit is all that is essential for exonuclease activity. One of the truncated but functional exonucleases (the pRAC3 exonuclease) has been purified and confirmed to be a 41-kDa polypeptide. The first 18 amino acids from the N terminus of the 41-kDa pRAC3 exonuclease were sequenced and fond to correspond to one of the translational start signals predicted from the nucleotide sequence of radC (Chu et al., in preparation).

Alleles↗

PubMed1986

Repair of heteroduplex plasmid DNA after transformation into Saccharomyces cerevisiae.

Purified heteroduplex plasmid DNAs containing 8- or 12-base-pair insertion mismatches or AC or CT substitution mismatches were used to transform Saccharomyces cerevisiae. Two insertion mismatches, separated by 943 base pairs, were repaired independently of each other at least 55% of the time. This suggested that repair tracts were frequently shorter than 1 kilobase. The two insertion mismatches were repaired with different efficiencies. Comparison of the repair efficiency of one mismatched site with or without an adjacent mismatch suggests that mismatches promote their own repair and can influence the repair of neighboring mismatches. When two different plasmids containing single-insertion mismatches were transformed into S. cerevisiae cells, a slight preference towards insertion was detected among repair products of one of the two plasmids, while no repair preference was detected among transformants with the second plasmid.

Base Sequence↗

PubMed1984

Origin, transmission, and segregation of mitochondrial DNA dimers in mouse hybrid and cybrid cell lines.

Hybrid and cybrid progeny lines were constructed from mouse LA9 cells which contain almost exclusively mtDNA monomers and LDTK cells which contain only unicircular mtDNA dimers. The proportion of mtDNA monomers and dimers in the progeny lines was determined both as a function of the number of population doublings since fusion and of selection for expression of a mutant phenotype encoded on one of the parental mtDNAs. There was no preferential segregation of either parental mtDNA in early-passage progeny lines, irrespective of whether or not selection was applied. In marked contrast, there was an accumulation of mtDNA dimers in late-passage progeny lines maintained in the absence of selection for a drug-resistance marker carried by the parental mtDNA monomers. When such selection was applied, roughly equal mass proportions of both parental mtDNAs were maintained in most lines. However, in several progeny lines, new types of mtDNA dimers carrying the selected resistance marker initially encoded in the monomeric mtDNA were present. In some of these latter lines, the new mtDNA dimers apparently arose from LA9 monomers, possibly by recombination. It is hypothesized that mammalian mitochondria normally have a recombination system which maintains low steady-state levels of mtDNA unicircular oligomers by preferentially resolving dimers into two monomers.

Animals↗

PubMed1975

Chloroplast DNA from higher plants replicates by both the Cairns and the rolling circle mechanism.

The chloroplast DNA (ctDNA) from pea and corn plants contains both Cairns type and rolling circle replicative intermediates. Denaturation mapping studies with pea ctDNA molecules have shown that the rolling circles initiate replication at or near the site where the Cairns replicative intermediates terminate replication. These results suggest that the rolling circles are initiated by a Cairns round of replication. A model for the replication of the chloroplast DNA is based on these results.

Chloroplasts↗

PubMed1975

Denaturation mapping studies on the circular chloroplast deoxyribonucleic acid from pea leaves.

The structure of circular pea chloroplast DNA (ctDNA) has been analyzed by denaturation mapping. All of the pea ctDNA molecules that were examined had identical gross base sequences. Denaturation maps were constructed at denaturation levels of 2.5%, 22%, and 44%. These denaturation maps showed that the circular pea ctDNA contained six small AT-rich regions on one-half of the DNA molecule, and two small GC-rich regions on the other half of the DNA molecule. The structure of pea ctDNA circular dimers was also examined. The results showed that the pea ctDNA circular dimers consisted of two monomer length units integrated in tandem repeat.

Chloroplasts↗

PubMedDate not supplied

Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro.

RecA-like activities that can form hybrid DNA in vitro have been identified in a wide variety of organisms. We have previously described the strand exchange protein 1 (SEP1) from the yeast Saccharomyces cerevisiae that can form hybrid DNA in vitro. Purified as an Mr 132,000 polypeptide, recent molecular and immunological studies have now shown that the native form is an Mr 175,000 polypeptide containing strand exchange activity. The gene encoding SEP1 has been cloned and sequenced. The primary sequence failed to reveal any significant sequence homology to other sequences in data base searches. In vivo SEP1 was found to be essential for normal meiosis as cells containing a homozygous insertion mutation in the SEP1 gene failed to sporulate. In order to identify additional factors that are involved in hybrid DNA formation in S cerevisiae, we used an in vitro stimulation assay to identify proteins that reconstitute strand exchange activity in reactions containing limiting amounts of SEP1. We have identified two proteins that functionally interact with SEP1. First, an Mr 34,000 single-stranded DNA binding protein stimulated the reaction by lowering the requirement for SEP1 about 3-4 fold. This protein is a fragment of the large subunit of a hetero-trimeric complex called yRP-A (yRF-A) which is thought to be the functional eukaryotic equivalent of single-stranded DNA binding proteins in prokaryotes. The gene encoding this protein (RPA1) is essential for growth. Second, an Mr 33,000 polypeptide, termed Stimulatory Factor 1 (SF1), dramatically stimulated the SEP1 catalyzed reaction by lowering the requirement for SEP1 about 300 fold.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA, Fungal↗

PubMedDate not supplied

The role of heteroduplex correction in gene conversion in Saccharomyces cerevisiae.

Two different models have been proposed to explain the relative frequencies of the non-mendelian allelic segregations which are detected by tetrad analysis after meiosis in fungi. The first model maintains that 6:2 type tetrads result from correction of heteroduplexes containing mismatched sites and 5:3 type tetrads result from failure to correct mismatched sites. The second model suggests that 6:2 segregations result from the filling-in of double-strand gaps using information obtained from both strands of a homologous duplex. In this model 5:3 type tetrads result if the allele is included in the heteroduplex regions flanking the gap and the resulting mismatched nucleotides are not corrected. We have studied the correction of heteroduplex plasmid DNA in pms1 mutant strains of Saccharomyces cerevisiae, which are known to exhibit higher frequencies of 5:3 type tetrads and lower frequencies of 6:2 tetrads than wild-type strains. Our results suggest that the pms1 mutation causes a defect in mismatch correction, supporting the hypothesis that meiotic gene conversion in wild-type yeast cells often results from the correction of heteroduplex DNA.

DNA Repair↗