Untersuchungen zur Funktion der Gene MPH1 und MMS2 aus Saccharomyces cerevisiae bei der fehlerfreien Umgehung von replikationsarretierenden DNA-Schäden

Abstract

in vivo error free bypass of replication blocking lesions is done by at least two distinct processes: homologous recombination via sister chromatid interaction (SCI) and post replicative repair (PRR). Additionally PRR is divided into an error free and an error prone sub branch. Homologous recombination requires the proteins Rad51 and Rad52. The error prone PRR is done via Rev3 and Rad30 dependant translesion synthesis, but the mechanism of error free PRR still remains unknown. However, polyubiquitylation of PCNA at lysine 164 in an Ubc13/Mms2 and Rad5 dependant manner seems to be a prerequisite for error free PRR and is done via linking of ubiquitin-internal lysine 63. In literature, postreplicative repair and homologous recombination are generally described as two processes, alternatively used by cells to bypass DNA-lesions. In contrast the data in this work shows, that both error free PRR and homologous recombination are in fact not independent of each other, but partially overlapping: o rad51 ist epistatic to mms2 with regards to sensitivity against 4-NQO. o rad51 ist epistatic to mms2 with regards to spontaneous mutation rates. o as in rad51-mutants, the rate of spontaneous sister chromatid interactions is slightly increased in mms2-mutants. o Frequency of 4-NQO-induced sister chromatid interactions is severely decreased in mms2- as well as in ubc13-mutants. o the phenotype of ubc13 can be complemented and depends on the catalytic function of Ubc13 protein. o Mutation of the ubiquitin internal lysine 63 causes a ubc13 like phenotype. o Mutation of the Mms2/Ubc13 substrate (PCNA-K164R) causes a similar phenotype as deletion of MMS2 and UBC13 and the mutation is epistatic to ubc13 with regards to sensitivity against 4-NQO. In this work, sister chromatid interaction have been detected via a system called “KanKanMX4” module, which was newly generated by the group of Dr. W. Kramer. With this module we measured the frequencies of G418-resistants generated via deletion of a direct repeat. The data acquired showed, that reversion of the G418 resistance is strictly dependant on homologous recombination: o rad52-mutants show neither spontaneous nor 4-NQO induced reversions of the KanKanMx4 module. o rad51-mutants show a severe reduction in 4-NQO induced reversion frequencies. o Spontaneous reversion rate in rad51 is increased, which is most likley the result of an increase of SSA events and a simultaneous decrease of SDSA and DSBR Through genetical interaction studies between rad30, rev3, rad51 and mms2 evidences for polyubiquitylation of PCNA being involved in Rev3-dependant mechanisms could be found. Also evidences for involvement of other, but still unkown mechanisms were found: o rad51 and rev3 are synergistic with regards to sensitivity against MMS, but additional deletion of mms2 doesn’t have any further impact on the sensitivity. o rad51rad30 and mms2 are additive to synergistic with regards to the sensitivity against UV-irradiation. mms2 and rad51rev3 in contrast are only subadditive. Due to this data, as the most likely model for a function of Mms2/Ubc13, a stimulation of Rev3 dependant processes as well as the stimulation of homologous recombination through direct interaction with polyubiquitylated PCNA was proposed. The data acquired in this work also showed, that the recently proposed function of Mph1 during cross over prevention might not be the only task of Mph1: o mph1 shows a reduction in camptothecin, MMS and 4-NQO induced reversion frequencies, with the most severe reduction at 4-NQO. o mph1 shows a similar increase in spontaneous reversion rate as rad51. o mph1 shows almost no reduction in the 4-NOQ induced reversion frequencies measured with the his3-5"/his3-3"-module (Fasullo & Davis, 1987, Proc Natl Acad Sci USA, 84, 6215-6219), which detects primarily cross over events.