| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
DNA polymerase ε (Pol ε) of Saccharomyces cerevisiae plays an essential role in the chromosomal DNA replication. The DNA polymerase activity of the enzyme resides in the N-terminal half of 256-kDa catalytic polypeptide encoded by Pol2 gene, and cells expressing a hampered Pol2 that lacks the N-terminal half can grow. On the other hand, the C-terminal half of Pol2 is essential for cell proliferation. Furthermore, genetical experiments suggested that the C-terminal half is involved in S-phase checkpoint control and initiation of DNA replication. To elucidate biochemical functions of these two domains of Pol2 and other auxiliary subunits of Pol ε, we have carried out the following biochemical studies.
1. PCNA-dependent DNA synthesis by Pot ε.
Pol ε is a four-subunit complex consisting of Pol2, Dpb2, Dpb3, and Dpb4, whereas p145 consists solely of a 145-kDa polypeptide conesponding to the N-terminal half of Pol2. Both Pol ε and p145 showed essentially the same ability to catalyze a highly pr
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ocessive DNA synthesis. Increasing the ionic strength in the reaction, the processivity of DNA synthesis decreased. Under these conditions, however, the processive DNA synthesis by Pol ε was fully recovered by the addition of PCNA and RF-C.The recovery of DNA synthesis by p145 was only about 10%. These results indicated that PCNA/RF-C-dependent DNA synthesis by Pol ε requires the C-terminal half of Pol2 and/or other subunits.
2. Double-stranded DNA binding capacity of Pol ε.
Using gel mobility shift assay, we have assessed DNA binding capacities of Pal ε and p145. It appeared that the C-terminal half of Pol2 and/or other subunits possess a novel biochemical function that facilitates strong association of Pol ε with double-stranded DNA.This DNA binding did not affect the DNA polymerase activity of the enzyme.
3. Expression of subunits of Pol ε in silkworm and purification of the subunits.
Genes encoding Pol ε subunits were cloned with a baculovirus vector and their products were searched in extracts of silkworm infected with the recombinants. Dpb2, Dpb3, and Dpb4 proteins were found to be expressed in the silkworm, but the expression of Pol2 was not successful. From the extracts, Dpb2 protein was purified to homogeneity Less
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