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Highly frequent frameshift DNA synthesis by human DNA polymerase mu
1Graduate Center for Toxicology, University of Kentucky, Lexington, Kentucky 40536, USA.
Abstract:
DNA polymerase mu (Polmu) is a newly identified member of the polymerase X family. The biological function of Polmu is not known, although it has been speculated that human Polmu may be a somatic hypermutation polymerase. To help understand the in vivo function of human Polmu, we have performed in vitro biochemical analyses of the purified polymerase. Unlike any other DNA polymerases studied thus far, human Polmu catalyzed frameshift DNA synthesis with an unprecedentedly high frequency. In the sequence contexts examined, -1 deletion occurred as the predominant DNA synthesis mechanism opposite the single-nucleotide repeat sequences AA, GG, TT, and CC in the template. Thus, the fidelity of DNA synthesis by human Polmu was largely dictated by the sequence context. Human Polmu was able to efficiently extend mismatched bases mainly by a frameshift synthesis mechanism. With the primer ends, containing up to four mismatches, examined, human Polmu effectively realigned the primer to achieve annealing with a microhomology region in the template several nucleotides downstream. As a result, human Polmu promoted microhomology search and microhomology pairing between the primer and the template strands of DNA. These results show that human Polmu is much more prone to cause frameshift mutations than base substitutions. The biochemical properties of human Polmu suggest a function in nonhomologous end joining and V(D)J recombination through its microhomology searching and pairing activities but do not support a function in somatic hypermutation.
Insights
DNA polymerase mu (Polmu) efficiently synthesizes DNA with frameshift mutations, particularly deletions, opposite repeat sequences. Its microhomology search and pairing suggest roles in DNA repair, not somatic hypermutation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA polymerase mu (Polmu) is a novel polymerase X family member.
- Its in vivo function remains largely unknown, with prior speculation suggesting a role in somatic hypermutation.
Purpose of the Study:
- To elucidate the in vivo function of human Polmu through in vitro biochemical analyses.
- To characterize the DNA synthesis fidelity and mechanisms of purified human Polmu.
Main Methods:
- Purification of human DNA polymerase mu.
- In vitro biochemical assays to analyze DNA synthesis activity and fidelity.
- Examination of DNA synthesis opposite various template sequences and mismatched bases.
Main Results:
- Human Polmu exhibits an exceptionally high frequency of frameshift DNA synthesis, primarily -1 deletions opposite single-nucleotide repeats.
- DNA synthesis fidelity is highly sequence-context dependent.
- Polmu efficiently realigns primers with microhomology regions in the template, facilitating microhomology search and pairing.
- Polmu is significantly more prone to frameshift mutations than base substitutions.
Conclusions:
- Human Polmu's biochemical properties suggest a role in nonhomologous end joining and V(D)J recombination due to its microhomology search and pairing capabilities.
- The findings do not support a role for Polmu in somatic hypermutation.
- Polmu's propensity for frameshift mutations highlights its unique DNA repair functions.