Defining error-free postreplication repair in Saccharomyces cerevisiae
Broomfield, Stacey D.
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The 'mms2-1' mutant was initially isolated by its sensitivity to the alkylating agent methyl methanesulfonate (MMS). I screened a yeast genomic library and identified the 'MMS2' gene. Disruption of 'MMS2' results in enhanced sensitivity to both MMS and UV and 'mms2-1' was found to be a missense mutation with partial loss of functions. Mms2 shares homology with ubiquitin-conjugating enzymes (Ubcs), but does not function as a typical Ubc in ubiquitination. The only other 'ubc' mutant that displays a UV and MMS sensitivity is 'ubc2' ('rad6') which is involved in postreplication repair (PRR) and mutagenesis. Epistatic analysis placed 'MMS2' within the 'RAD6' pathway but is parallel to the error-prone PRR subpathway consisting of 'REV1, REV3' and 'REV7 '. Mms2 is defined as a member of error-free PRR, since 'mms2 '[Delta] cells display an increase in the spontaneous and UV-induced mutations, and these phenotypes depend on 'REV3. mms2'[Delta] and 'rev3'[Delta] are synergistic with respect to killing by DNA damaging agents, indicating that they function in alternative pathways. Study of 'rad6'[Delta]'1-9, po130-46, po13-13, rad5'[Delta] and 'rad30'[Delta] alleles has placed these genes in the error-free PRR pathway. I found that 'rad6'[Delta]' 1-9' is epistatic to 'mms2'[Delta], while ' pol30-46, rad5'[Delta] and 'rad30'[Delta] all display an additive effect with 'mms2'. A 'srs2'[Delta] mutation is able to rescue the UV sensitivities of the 'rad6'[Delta] and 'rad18'[Delta] mutants, and is epistatic to 'mms2 '[Delta] and 'rev3'[Delta]. Furthermore, 'srs2 '[Delta] suppresses the MMS sensitivity of 'pol30-46' and 'rad5'[Delta], indicating that Srs2 controls the entire PRR pathway. In addition to PRR, the Ubc activity of Rad6 is involved in other cellular functions including sporulation, telomere silencing and protein degradation. The 'mms2'[Delta] mutation results in moderate defects in sporulation and protein degradation but has no effect on telomere silencing. This indicates that Mms2 may act as an accessory protein for Rad6, or alternatively, may provide an similar activity via a pathway distinct from Rad6. Two human Mms2 homologues, 'hMMS2' and 'CROC1', have been isolated. 'hMMS2' can suppress the yeast ' mms2'[Delta] UV and MMS sensitivity and elevated mutation rate. Further analysis is required to assess whether 'hMMS2' and ' CROC1' possess analogous activities in mammalian cells.
DegreeDoctor of Philosophy (Ph.D.)
DepartmentMicrobiology and Immunology
ProgramMicrobiology and Immunology
Copyright DateDecember 2000