8 research outputs found
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Modulation of deoxyribonucleoside triphosphate levels, DNA synthesis rates and fidelity in mammalian cells
Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in cells. In an in vitro replication system involving semiconservative replication from an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human cell extracts in reaction mixtures containing "biologically biased" dNTP pools estimated from HeLa cell nuclei is not significantly different from that seen when replication is done with equimolar dNTP concentrations. Significant reduction of dGTP pool while keeping other dNTPs at "biologically biased" dNTP concentrations during replication reaction also did not increase mutation frequency. In contrast, in vitro replication with dNTP concentrations calculated from normal diploid fibroblast cells, which are three- to four-fold lower in concentrations, showed a marked reduction of the observed mutation frequency, showing the importance of overall dNTP levels during replication on mutation frequency in vitro. When whole-cell dNTP concentrations in HeLa cells were measured during S-phase, dNTP levels underwent a transient decrease in the middle of S-phase. Average HeLa cells' dNTP levels were also found to correlate with average DNA accumulation rates during S-phase, although no detailed relationship can yet be deducted from the available data. No significant changes in the ratio of the four dNTP concentrations were found during S-phase. Mutation rates of green fluorescent protein (GFP) inserted in either middle or late-replicating region of a chromosome in HeLa cells also correspond to average DNA accumulation rates and dNTP levels during middle and late S-phase. The late-replicating GFP-HeLa cells have a higher mutation rate than the middle-replicating GFP-HeLa cells, as the average DNA accumulation rates and dNTP pool levels were also lower in the middle compared to late S-phase. Taken together, these observations indicate that dNTP levels could play a role in determining the S-phase DNA replication rate and also the replication fidelity in mammalian cells
A Role for Msh6 But Not Msh3 in Somatic Hypermutation and Class Switch Recombination
Somatic hypermutation is initiated by activation-induced cytidine deaminase (AID), and occurs in several kilobases of DNA around rearranged immunoglobulin variable (V) genes and switch (S) sites before constant genes. AID deaminates cytosine to uracil, which can produce mutations of C:G nucleotide pairs, and the mismatch repair protein Msh2 participates in generating substitutions of downstream A:T pairs. Msh2 is always found as a heterodimer with either Msh3 or Msh6, so it is important to know which one is involved. Therefore, we sequenced V and S regions from Msh3- and Msh6-deficient mice and compared mutations to those from wild-type mice. Msh6-deficient mice had fewer substitutions of A and T bases in both regions and reduced heavy chain class switching, whereas Msh3-deficient mice had normal antibody responses. This establishes a role for the Msh2-Msh6 heterodimer in hypermutation and switch recombination. When the positions of mutation were mapped, several focused peaks were found in Msh6β/β clones, whereas mutations were dispersed in Msh3β/β and wild-type clones. The peaks occurred at either G or C in WGCW motifs (W = A or T), indicating that C was mutated on both DNA strands. This suggests that AID has limited entry points into V and S regions in vivo, and subsequent mutation requires Msh2-Msh6 and DNA polymerase
MSH2βMSH6 stimulates DNA polymerase Ξ·, suggesting a role for A:T mutations in antibody genes
Activation-induced cytidine deaminase deaminates cytosine to uracil (dU) in DNA, which leads to mutations at C:G basepairs in immunoglobulin genes during somatic hypermutation. The mechanism that generates mutations at A:T basepairs, however, remains unclear. It appears to require the MSH2βMSH6 mismatch repair heterodimer and DNA polymerase (pol) Ξ·, as mutations of A:T are decreased in mice and humans lacking these proteins. Here, we demonstrate that these proteins interact physically and functionally. First, we show that MSH2βMSH6 binds to a U:G mismatch but not to other DNA intermediates produced during base excision repair of dUs, including an abasic site and a deoxyribose phosphate group. Second, MSH2 binds to pol Ξ· in solution, and endogenous MSH2 associates with the pol in cell extracts. Third, MSH2βMSH6 stimulates the catalytic activity of pol Ξ· in vitro. These observations suggest that the interaction between MSH2βMSH6 and DNA pol Ξ· stimulates synthesis of mutations at bases located downstream of the initial dU lesion, including A:T pairs