Untersuchungen zur Rolle des <i>MPH1</i>-Gens aus <i>Saccharomyces cerevisiae</i> bei der Reinitiation der Replikation nach schadensinduzierten Arresten

Abstract

For the error-free removal of DNA damages the damaged bases are usually removed by mechanisms like nucleotide or base excision repair (NER, BER), mismatch repair etc. and the resulting gap is subsequently filled by a DNA polymerase. A prerequisite for these repair mechanisms is the availability of the information on the intact opposite strand. However, during replication the parental strands are separated and therefore repair by mechanisms like NER or BER cannot take place. If a lesion leads to an arrest of the replicative polymerase in E. coli and replication cannot be reinitiated, the cell will die.One possibility for an error-free reinitiation of replication are mechanisms using homologous recombination. By using the information of the sister chromatid for DNA synthesis, lesions can be bypassed in an error-free manner. Having created a 3 -end downstream the lesion the replication can be reinitiated, using this 3 -end as a primer. The lesion can subsequently be removed by repair pathways like NER or BER.The MPH1 gene from Saccharomyces cerevisiae is involved in a recombinogenic pathway for the error-free bypass of DNA lesions which can be bypassed in the absence of Mph1 by translesion polymerases. Therefore we used an assay for detection of spontaneous recombination events in the cell. Additionally, it was possible to measure the influence of the DNA-damaging agents 4-NQO and camptothecin on recombinogenic events.With this assay the influence of mutations in mph1, rev3, rad30, sgs1 and srs2 on recombination events induced by 4-NQO and camptothecin were determined and compared with spontaneous and 4-NQO induced mutations in the canavanine forward mutation system. Additionally, the effect of a heterologously expressed E. coli recG on repeat deletions, mutations and DNA damage sensitivities was measured. The data support the idea of an involvement of Mph1 in a recombinogenic pathway for rescue of arrested replication forks which are mainly stalled at structures which block the replicative polymerases. Possible roles of the other investigated genes in reinitiation and repair mechanisms are discussed.