DNA damage could be repaired correctly or sometimes misrepaired to produce GCRs. Because of the complexity for choice of pathways to deal with DNA damage, cells need to have mechanisms to promote the most appropriate repair pathway. Our results suggest that Mph1p can promote a GCR pathway by partially suppressing HR.
Mph1p enhances GCRs by partially compromising HR and activating a GCR pathway ( and ). The suppression of HR by Mph1p is likely achieved by stabilizing RPA () binding of DNA, thereby blocking Rad52p-mediated Rad51p nucleofilament formation (). Consistent with these ideas, complete HR inactivation allowed excess Mph1p to promote GCR more efficiently (). The observation that Rad52p overexpression, but not that of Rad51p and Rad54p, could reduce Mph1p-induced GCRs ( and not depicted) also suggests that Mph1p suppresses HR before Rad51p recruitment to the DSB. Notably, Mph1p was captured by affinity capture mass spectrometry using RPA as bait (Gavin et al., 2006
), and physically interacted with RPA (). Collectively, we propose that Mph1p interacts with and stabilizes RPA-coated single-stranded DNA, and this prevents Rad52p-mediated Rad51p nucleofilament formation. This role of Mph1p is further supported by the observation that the mph1Δ
mutation reduces the number of cells producing spontaneous or DNA damage–induced RPA foci ( and not depicted).
Alternatively, it is possible that excess Mph1p could interfere with RPA or Rad51p-Rad52p recruitment to DNA damage by scavenging them. Nevertheless, there were no physical interactions between Mph1p and Rad52p or between Mph1p and Rad51p (unpublished data). Therefore, GCRs promoted by Mph1p are likely caused by the blocking of Rad51p-Rad52p through its interaction with RPA.
GCRs enhanced by excess Mph1p could be driven by the interference of DNA replication through its interaction with RPA. Even though excess Mph1p did not cause a significant change in the proportion of cells in S phase (unpublished data), we cannot rule out the possibility that the Mph1p-induced GCR enhancement arises when excess Mph1p perturbs DNA replication in the small proportion of cells that are not detectable by FACS analysis.
The GCR-promoting activity by Mph1p is required for GCRs produced in strains having rad5Δ, rad18Δ, mec1Δ, or rfa1-t33 mutations under physiological conditions (). For its GCR-promoting activity, Mph1p's interaction with RPA seems to be essential; in contrast, the helicase activity of Mph1p is only partially required (). The blocking of Rad51p filament formation by Mph1p is solely dependent on its interaction with RPA, not its helicase activity ( and ). Finally, because the motif mutants of Mph1 could still rescue the MMS sensitivity of mph1Δ (), only the loss of Mph1's GCR-promoting activity (i.e., its interaction with RPA) results in the synergistic sensitivity to MMS with the srs2Δ mutation. Thus, the RPA interaction seems to be essential for both the GCR-promoting activity and the Srs2p-like repair functions of Mph1p.
Even though excess Mph1p increased GCRs by partially suppressing HR, the complete inactivation of HR does not increase GCR when Mph1p is expressed in physiological conditions, except the rad52Δ
mutation that also inactivates the break-induced replication that is important to suppress GCRs (Myung et al., 2001a
). Therefore, partial HR activity is necessary to promote GCR, at least when Mph1p is expressed in physiological conditions. The requirement of partial HR activity for GCR formation is further supported by the suppression of GCRs in the rad5Δ
strain by the inactivation of HR (Motegi et al., 2006
). This partial HR activity could be required to process DNA damage to produce intermediates, presumably DSB, for GCR formation. However, such activity might not be required if excess Mph1p covers RPA-coated single-stranded DNA and causes a break in the DNA. Alternatively, partial HR activity might allow GCR machinery to access DNA damage, whereas excess Mph1p could simply overcome such a requirement by blocking the access of other repair proteins.
One unique feature of Mph1p discovered in this study is the demonstration of its role in suppressing HR. Even though there are several studies that suggest that mph1Δ
is epistatic to mutations in HR genes (Scheller et al., 2000
; Prakash et al., 2005
; Onge et al., 2007
), the mph1Δ
mutation did not change the HR rate (unpublished data). No change in the HR rate by the mph1Δ
mutation could be caused by the activation of postreplication repair by the mph1Δ
mutation (Scheller et al., 2000
). Elevated postreplication repair could bypass damaged DNA before HR repairs it in the mph1Δ
strain, resulting in no change of the HR rate. Alternatively, Srs2p could suppress HR in the absence of Mph1p, which is supported by synergistic sensitivity to MMS by mph1Δ
Even though Srs2p could function similarly to Mph1p to promote GCR (Motegi et al., 2006
), we did not detect GCR enhancement under the same expression system with Srs2p (unpublished data). This may be caused by the toxicity of Srs2p overexpression, which has been observed in a yeast Srs2p purification study (Krejci et al., 2003
When cells reach late S or G2 phase, telomerase activity is high to replicate the end of chromosome (Marcand et al., 2000
). This telomerase activity seems to promote de novo telomere addition–type GCRs. Excess Mph1p could augment GCRs from DNA damage by partially suppressing HR at the stalled replication forks. This sustained replication stall may lead to DSBs, thus providing substrates for active telomerase to carry out de novo telomere addition (the major type of GCR observed in this study).
Multiple choices to repair DNA lesions during DNA replication could result in different outcomes. Usually, these outcomes are beneficial for cells, but sometimes they can result in harmful mutations. In the present study, we uncovered Mph1p as an important decision maker between HR and GCR. The abnormal expression or mutation of MPH1
can lead to undesirable outcomes, like GCRs (Mph1p overexpression) or mutations (mph1Δ
; Scheller et al., 2000
). Mph1p's putative human homologue FANCM could have a similar function for directing different DNA repair pathways. Therefore, the cancer predisposition observed in FA patients could be caused by erroneous repair choice.