DNA repair by polymerase delta in Saccharomyces cerevisiae is not controlled by the proliferating cell nuclear antigen-like Rad17/Mec3/Ddc1complex

DNA damage activates several mechanisms such as DNA repair and cell cycle checkpoints. The Saccharomyces cerevisiae heterotrimeric checkpoint clamp consisting of the Rad17, Mec3 and Ddc1 subunits is an early response factor to DNA damage and activates checkpoints. This complex is structurally similar to the proliferating cell nuclear antigen (PCNA), which serves as a sliding clamp platform for DNA replication. Growing evidence suggests that PCNA-like complexes play a major role in DNA repair as they have been shown to interact with and stimulate several proteins, including specialized DNA polymerases. With the aim of extending our knowledge concerning the link between checkpoint activation and DNA repair, we tested the possibility of a functional interaction between the Rad17/Mec3/Ddc1 complex and the replicative DNA polymerases alpha, delta and epsilon. The analysis of sensitivity response of single and double mutants to UVC and 8-MOP + UVA-induced DNA damage suggests that the PCNA-like component Mec3p of S. cerevisiae neither relies on nor competes with the third subunit of DNA polymerase delta, Pol32p, for lesion removal. No enhanced sensitivity was observed when inactivating components of DNA polymerases alpha and epsilon in the absence of Mec3p. The hypersensitivity of pol32delta to photoactivated 8-MOP suggests that the replicative DNA polymerase delta also participates in the repair of mono- and bi-functional DNA adducts. Repair of UVC and 8-MOP + UVA-induced DNA damage via polymerase delta thus occurs independent of the Rad17/Mec3/Ddc1 checkpoint clamp.

Structure of rotavirus particle: interaction of the inner capsid protein VP6 with the core polypeptide VP3

The structural relationship between VP6 (inner capsid polypeptide) and the viral core was studied using chemical cross-linking with dithiobis(succinimidyl propionate). Crosslinked single shelled and reconstituted rotavirus particles, suggest the existence of a complex organization of VP6 molecules in the inner capsid and a direct interaction with the core polypeptide VP3. The inhibition of the recovery of RNA polymerase activity associated with the reconstitution of the single shelled particle in the presence of antiVP6 monoclonal antibodies indicates that a VP6 domain between amino acids 56 and 58 seems to be important in viral transcription. A VP6 gene temperature-sensitive mutant (ts G) carrying a mutation affecting assembly of single shelled particles was used in reconstitution experiments. The mutant was able to recover RNA polymerase activity at restrictive temperature. Wild type cores or VP6 were able toreconstitute the particle with both the mutant cores and VP6. These results suggest the existence of various steps for the assembly of single shelled particles, where the VP6-VP3 interaction seems to be important for recovery of RNA polymerase activity

Changes in DNase and DNA-polymerase activities in different tissues of chick with age.

The presence of acid and alkaline DNases in nuclei of chick brain cells has been demonstrated. The activities of these two DNases along with those of DNA-polymerases were assessed in chicken tissues with known varied cell proliferative capacities (eg., spleen, kidney and brain) at different ages. The results indicate that the acid and alkaline DNases are probably the 'house keeping' enzymes with a constitutive role in DNA repair process, the former with a DNA-repair process that is linked to cell proliferation (DNA synthesis) while the latter with the basal DNA-repair operations that must go on at all times without any regard to the cell division process. Chicken brain, unlike that of rat, possesses significant levels of aphidicolin sensitive DNA-polymerase(s) that are considered to be more replication oriented enzymes suggesting that the replication potential of adult and old avian brain cells may be different from that of a mammalian brain.

Serological pattern of hepatitis B virus markers (HBsAg, anti-HBs, IgM anti-HBc and HBV specific DNA polymerase) in leprosy patients.

Sera of 134 lepromatous (LL/BL) and 57 tuberculoid (TT/BT) leprosy patients were analysed for four HBV markers. HBsAg was detected in 6.71% of lepromatous and 3.5% of tuberculoid sera. The per cent positivity of lepromatous and tuberculoid sera for anti-HBs antibodies was 30.59% and 35.08%, respectively. The positivity of normal sera for HBsAg and anti-HBs was 3.60% and 21.69%, respectively. The difference in the positivity of three groups of sera (lepromatous, tuberculoid and normal) for HBsAg or anti-HBs was not statistically significant. Anti-HBc (IgM) antibodies were detected in 6% of lepromatous sera. HBV-specific DNA-polymerase activity was found in 22.22% of HBsAg positive (but anti-HBc negative) sera, and 66.66% of anti-HBc positive (but HBsAg negative) sera. The pattern of acute HBV infection in leprosy patients followed the typical pattern prevalent in the normal population.

Properties of Hepatitis B Virus Associated DNA Polymerase

The nature of hepatitis B virus (HBV) particle associated DNA polymerase was studied in relation to various enzyme inhibitors including antiviral agents. HBV DNA polymerase required high concentration of MgCl2(> 30 mM) and neutral pH for its full activity. p-chloromercuribenzoate was a strong inhibitor (85% inhibition at 1 mM) but N-ethylmaleimide had much less inhibitory effect (20% inhibition at 10 mM). Phosphonoformic acid showed the greatest inhibitory effect on HBV-DNA polymerase (almost complete inhibition at 100 microM) among phosphocompounds tested. Adenine arabinoside triphosphate (ara-ATP) and cytosine arabinoside triphosphate (ara-CTP) were competitive inhibitors with respect to their respective deoxyribonucleoside triphosphate (dATP and dCTP). Ara-CPT was a stronger inhibitor of HBV-DNA polymerase compared to ara-ATP. Ki values for ara-ATP and ara-CTP were 15.0 microM and 11.7 microM , respectively. HBV-DNA polymerase is characteristic in its ionic requirements and susceptibilities to certain inhibitors.