No abstract

Mutation of predicted 3′‐‐>5′ exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease‐deficient mutant pol3‐01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3‐01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3‐01/pol3‐01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 × 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3‐01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co‐regulated with these genes. We propose that the POL3 3′‐‐>5′ exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ‐‐>mutHLS pathway of DNA replication error correction in Escherichia coli.

DNA polymerases II (epsilon) and III (delta) are the only nuclear DNA polymerases known to possess an intrinsic 3'-->5' exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase delta and epsilon 3'-->5' exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 10(2) and 10(1), respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 x 10(3)-fold. Analysis of mutation rates of double mutants indicated that the 3'-->5' exonucleases of DNA polymerases delta and epsilon can act competitively and that, like the 3'-->5' exonuclease of DNA polymerase delta, the 3'-->5' exonuclease of DNA polymerase epsilon acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3'-->5' exonucleases of DNA polymerases delta and epsilon act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.

We have identified an amino-proximal sequence motif, Phe-Asp-lle-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3' --5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolinsensitive DNA polymerases possessing 3' -*5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase Il had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3' -* 5' exonuclease active site ofE. coiU DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3' -5' proofreading exonuclease activity. None ofthese residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila a DNA polymerases.Class B DNA polymerasest are related through a series of conserved amino acid sequences that occur in the order IV, II, VI, III, I, V, with region I being the most highly conserved (2). Regions II, III, I, and V are implicated in DNA polymerase functions (3)(4)(5)(6)(7)(8). The location of the 3' -* 5' exonuclease activity domain has been sought through amino acid sequence similarity with the apparently unrelated E. coli DNA polymerase I. Crystallographic and mutational evidence has determined that the 3' -*5' exonuclease active site of E. coli DNA polymerase I is composed of a group of carboxylate residues (D355, E357, D424, and D501) clustering around two metal ions that coordinate the 3'-terminal phosphate and a second group of residues (L361, F473, and Y497) located around the terminal base and ribose positions (9, 10). It has been proposed that residues D355 and E357 are conserved in the left part of region IV ofaphidicolin-sensitive DNA polymerases (11-13). Similarly, residues D424, Y497, and D501 were suggested to be conserved in the right part of, and distal to, region IV (4,12,13). Mutation to alanine of three of the relevant carboxylate residues in bacteriophage 029 DNA polymerase leads to exonuclease-deficient DNA polymerase activity (13). Though related to other aphidicolinsensitive DNA polymerases, 429 DNA polymerase does not have a well-conserved region IV (2) and may not be a good test case for alignments involving this region. With coliphage T4 DNA polymerase, which does have a well-conserved region IV (2, 11, 12), mutation to alanine ofresidues D189 and E191, predicted to correspond to the E. coli polymerase residues D355 and E357, does not lead to exonuclease deficiency, calling into question the concept of a generally conserved 3'-* 5' exonuclease active sit...

RA-related work-disability rates were similar in the USA and European countries. An apparent decrease in the prevalence of RA-related work disability since the 1970s may be related to a decrease in physically demanding work rather than to epidemiologic changes in RA. The majority of the literature addresses permanent disability and temporary work loss; none of the studies reviewed reported the effect of RA on presenteeism, i.e. work limitation from the employer perspective, and there are few published studies of the effectiveness of disease-modifying anti-rheumatic drugs in reducing work-related productivity loss.