RNA priming coupled with DNA synthesis on natural template by XXXX thymus DNA polymerase alpha-primase.

@entity10043 @entity70093 @entity6245 @entity21443 @entity71365 @entity291 @entity7391
A @entity291 genomic DNA library was surveyed with respect to the template activity for RNA-primed DNA synthesis by @entity291 thymus DNA polymerase alpha-primase complex. About 7% of the single-stranded DNA clones contained distinct initiation sites consisting of @entity7391 clusters of @entity7391 -rich sequences. The initiation sites were located at or near the 3'-end of the @entity7391 clusters. One of these sequences, containing a 10-mer @entity7391 cluster with major initiation sites, was analyzed in detail. By the successive substitutions of @entity6245 in the cluster with oligodeoxydenylate @entity21443 in the 5' to 3' direction, the minimum length of the initiation sequence was estimated to be as long as the 7-mer. In contrast, when one or three @entity6245 at the 3'-end of the cluster were replaced with (dA)n, the priming activity was largely lost, indicating that these @entity7391 residues were indispensable for priming. Furthermore, base substitutions of upstream or downstream sequences outside the @entity7391 cluster also decreased the total priming frequencies. Interestingly, the base substitutions inside or outside of the @entity7391 cluster sometimes caused a shift in the major priming sites. These results indicate that the minimum priming unit of the CTPPS1 template for RNA-primed DNA synthesis consists of a @entity7391 cluster (6-mer) with one @entity10043 at its 3'-border and that both the 3'-downstream 6-bases and the 5'-upstream 17-bases modulate the priming by enhancing the priming frequency and/or slightly shifting the sites of initiation of primer synthesis. It was also revealed that the lengths of the product RNA primers became shorter as the length of @entity7391 cluster was shortened by substitution with (dA)n. The gel retardation assay further showed that the complex formation between DNA polymerase alpha-primase and the DNA templates was strongly in competition with @entity70093 , @entity71365 , and poly(dT) but not with poly(dA). Furthermore, template activities as well as the @entity7391 contents of a series of base-substituted DNA correlated well with their affinities to the enzyme, as measured by both gel retardation assay and their Km values for the priming reaction. Apparently, DNA polymerase alpha-primase primarily recognizes the minimum priming unit consisting of a @entity7391 cluster with a @entity10043 at the 3'-boundary of @entity10043 , but the initiation of primer RNA synthesis can be modified by @entity7391 residues outside of the minimum priming unit.
