DNA replication mechanisms leading to """"""""non-semiconservative replication"""""""" have long been considered to play a role in genome instability in both prokaryotes and eukaryotes. Early studies of DNA polymerases revealed that these enzymes are capable of allowing primer slippage, such that long products with expanded simple direct repeat motifs are produced. Similarly, deletions, insertions, and mismatches are produced by a variety of DNA polymerases due to primer relocation mechanisms. Primer relocation usually involves """"""""jumping"""""""" of a nascent primer to a complementary sequence different from that in normal semiconservative replication. Molecular events such as these during DNA replication and repair are likely to be implicated in expansion of the triplet direct repeat (CTG)n in muscular dystrophy and Kennedy's disease and of the (CGG)n repeat in Fragile X Syndrome. Our hypothesis is that templating for DNA polymerases by sequences containing simple direct repeats leads to repeat expansion by virtue of the primer relocation mechanism. Our goal will be to duplicate DNA replication of triplet repeat sequences in vitro and then to study requirements for repeat instability.
The Specific Aims of this project are; 1) using purified DNA polymerases, examine the frequency of DNA replication frameshift errors during replication of a simple direct repeat sequence of various lengths: 2) using a crude nuclear extract system, examine the frequency of DNA replication frameshift errors for plasmid- based SV40 ori-dependent replication of a simple direct repeat sequence, as leading and lagging strand templates; 3) using a crude nuclear extract system for DNA repair, determine the sequence of newly synthesized products of repair of triplet repeat sequences with a single G-U mismatch; 4) to characterize in vitro replication by purified DNA polymerases on strategic single-stranded and double-stranded templates, as a function of the length and type of triplet repeat; 5) to test the hypothesis that DNA polymerase binding to the nascent primer template complex mediates primer relocation: We will study as a function of triplet repeats, binding constants for enzyme plus template primer interaction, pocessivity during replication, and polymerase-induced """"""""breathing"""""""" at the 3' end of the repeat-containing primer.
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