Identification of MHF1 and MHF2 as integral components of the FA core complex
To search for additional components of the FA core complex, we fractionated HeLa nuclear extract by gel-filtration chromatography, collected peak fractions containing FANCM (peak 1 in ), and immunoprecipitated with a FANCM antibody. Silver-staining (), mass spectrometry analyses (data not shown), and immunoblotting () revealed the presence of all known components of the FA core complex (FANC-A,-B, -C, -E, -F, -G -L, -M, FAAP100 and FAAP24) in the FANCM immunoprecipitate. Most of the Bloom syndrome complex components (BLM, TOPIIIα, RMI1/BLAP75, RPA70, and RPA32) were also identified by mass spectrometry (data not shown), supporting our previous findings that the FA core and BLM complexes associate in a super-complex, BRAFT (Wang, 2007
Identification of MHF1 and MHF2 as integral components of the FA core complex
Silver-staining detected two polypeptides of about 16 and 10 kDa that were not previously identified (). Mass spectrometry revealed the 16 kDa protein as CENP-S or APITD1 (gene accession ID: NP_954988) (Foltz et al., 2006
; Krona et al., 2004
), and the 10 kDa polypeptide as a protein with accession ID of A8MT69 or CENP-X (Amano et al., 2009
). A8MT69 has a confusing name of STRA13, which refers to two distinct proteins: one is A8MT69, and the other is an unrelated helix-loop-helix transcription factor. Of 56 published articles on STRA13, only one is on A8MT69, which shows that deletion of the A8MT69 ortholog in fission yeast may result in sensitivity to several genotoxins (Deshpande et al., 2009
). To avoid the confusion, we renamed the 16 and 10 kDa polypeptides as MHF1 and MHF2, respectively (for FANCM
old protein 1
The following evidence indicates that MHF1 and MHF2 are components of the FA core complex. First, immunoblotting showed that antibody against MHF1 or MHF2 recognized the corresponding polypeptide in the immunoprecipitate obtained with either FANCM or FANCA antibody (). Second, one peak of MHF1 or MHF2 on Superose 6 column is coincident with that of FANCM, supporting the notion that they are present in the same FA core complex (, peak 1). Third, reciprocal immunoprecipitation using a MHF1 antibody from the pooled Superose fractions of peak 1 obtained the same set of polypeptides isolated by the FANCM antibody, including components of both FA core and BLM complexes, as evidenced by silver-staining (), mass spectrometry (data not shown), and immunoblotting (). Finally, MHF2-associated polypeptides isolated by a Flag antibody from the extract of HeLa cells stably expressing Flag-tagged MHF2 also contained MHF1, FANCM and other FA core complex components ().
We noticed that MHF1 and MHF2 co-fractionate in two peaks on a Superose 6 column (). While peak 1 corresponds to the FA core complex of about 1 MDa, peak 2 corresponds to a complex of much smaller size. When peak 2 fractions were immunoprecipitated with a MHF1 antibody, only MHF1 and MHF2 were isolated (), as revealed by mass spectrometry (data not shown), indicating that these two proteins could comprise a complex distinct from the FA core complex. The two MHF proteins appear to be approximately equimolar amounts on the silver-stained gel, suggesting that they are likely obligate partners. Direct interaction between MHF1 and MHF2 was observed by mammalian two-hybrid analyses (Figure S1
MHF1 and MHF2 are conserved from human to yeast and form a new histone-fold complex
Bioinformatics revealed that both MHF proteins contain a histone-fold, which can mediate both protein-protein and protein-DNA interactions (Figure S2A and B
) (Arents and Moudrianakis, 1993
). Proteins containing this motif often associate to form heterodimeric or heterotetrameric complexes that bind to bent DNA. Our purification of MHF1 and MHF2 as a stoichiometric complex from HeLa extract () fits well with the bioinformatic prediction that they form a histone-fold complex.
We co-expressed MHF1 with HIS-tagged MHF2 in E. coli, and purified the complex using Talon metal affinity chromatography. Coomassie-staining revealed that the two proteins were present in approximately equimolar amounts (, lane 3), indicating that they indeed form a heterodimeric complex. We named this complex MHF.
MHF binds double-strand and branch-structured DNAs, but not single-strand DNA
MHF possesses DNA binding properties distinct from FANCM and FAAP24
Like other histone-fold complexes, MHF was found to bind double-strand DNA (dsDNA), but not single-strand DNA (ssDNA) (, lanes 1-10). This activity requires the MHF complex because individual MHF subunit lacked the activity.
MHF was also found to bind several structured DNAs containing different branch points (, lanes 11-30). Notably, the affinity of MHF to these structures was either similar or somewhat reduced compared to that of dsDNA of the same length (compare lanes 10, 15, 20, 25 and 30), indicating that MHF has no increased affinity for branched DNA, and its binding may even be precluded by such structures. The binding of MHF to dsDNA was further visualized by electron microscopy (Figure S3A and B
). MHF appeared to form clusters which result in compaction of DNA, suggesting self-association between MHF proteins.
The DNA binding characteristics of MHF differ from those of FANCM and FAAP24, which specifically recognize branched and ssDNA, respectively (Ciccia et al., 2007
; Gari et al., 2008b
; Xue et al., 2008
). We propose that these proteins constitute a molecular machine that binds cooperatively to different parts of a stalled replication fork: FANCM binds the branch point, whereas MHF and FAAP24 associate with dsDNA and ssDNA regions, respectively (see Discussion). Moreover, we have also shown that MHF can bind chromatin (Figure S3C
) and cooperate with histone octamers to assemble into nucleosome structures in vitro (Figure S3D and E
), which is consistent with MHF aiding FANCM association with DNA in vivo. Furthermore, MHF can efficiently anneal complementary single-stranded DNAs (albeit not when they are pre-bound by RPA) (Figure S3F and G
), which could assist the catalysis of branch point migration by FANCM.
MHF and FANCM co-evolved and form an independent complex
Bioinformatics analyses revealed that MHF1 and MHF2 orthologs are present in all eukaryotes, including yeast (Figure S2C and D
). This feature is shared by FANCM but not by other FANC proteins or FAAPs, most of which have orthologs only in vertebrates (Wang, 2007
). The data suggest that FANCM, MHF1 and MHF2 may perform functions important to all eukaryotes that favor their co-evolution.
The co-evolution of FANCM and the two MHF proteins implies that they may constitute a complex that lacks other FANC proteins. To distinguish this complex from the FA core complex in HeLa cells, we omitted the Superose 6 fractionation step that was used for enrichment and purification of the FA core complex (). We performed immunoprecipitation directly from HeLa extract with the same MHF1 antibody, and obtained FANCM, MHF1 and MHF2 as the only three major polypeptides (). Other FANC proteins can only be detected by immunoblotting due to their lower levels (data not shown). The data suggest that significant amounts of MHF and FANCM are present in a complex largely free of other FANC proteins. We named this complex FANCM-MHF.
MHF stimulates DNA binding and fork reversal activities of FANCM
Majority of MHF and FANCM do not associate with the FA core complex
We quantitatively immunodepleted the FA core complex from HeLa extract with the FANCA antibody, and found that less than 30% of FANCM, MHF, and FAAP24 were co-depleted (Figure S4A
), Similarly, depletion of FAAP24 or MHF from the extract co-depleted FANCA by less than 25% (Figure S4B and C
). These data suggest that majority of FANCM and its two partners do not associate with the FA core complex. Conversely, immunodepletion of either FAAP24 or MHF co-depleted FANCM by about 70% and 85% (Figure S4B and C
), indicating that most of FANCM in cells associates with either or both of its partners.
MHF and FAAP24 can form separate complexes with FANCM
We immunoprecipitated MHF from the FAAP24-depleted extract and obtained FANCM but no FAAP24 (Figure S4B
, lane 4), suggesting that MHF and FANCM can form a complex without FAAP24. Similarly, we immunoprecipitated FAAP24 from the MHF-depleted extract and obtained FANCM but no MHF (Figure S4C
, lane 4), indicating FAAP24 and FANCM can also form a complex without MHF. Together with the results in which showed that FANCM and both partners co-IP, our data suggest that FANCM can associate with its partners in a combinatorial manner to form distinct complexes: FANCM-MHF, FANCM-FAAP24, and FANCM-MHF-FAAP24.
MHF interacts with FANCM and promotes its DNA binding activity
We mapped the MHF-interaction domain within FANCM to a region near the helicase domain of FANCM (aa. 661-800) by IP-Western analyses of a series of FANCM deletion mutants ( and S4D
). We have previously shown that both the full-length FANCM protein and its N-terminal fragment (1-754 aa.) encompassing the helicase domain (FANCM754
) have high affinity for branched DNA structures, but not for dsDNA (Gari et al., 2008b
; Xue et al., 2008
). The current findings that MHF interacts with both FANCM and dsDNA predict that MHF may recruit FANCM to dsDNA. Consistent with this prediction, FANCM754
alone exhibited little dsDNA binding activity (, lanes 2-4), while MHF and FANCM754
together displayed strong binding activity (see the supershifted band in , lanes 6 to 8 vs. 5).
FANCM and MHF bind DNA synergistically
The fact that FANCM and the two MHF proteins can form a complex raised a possibility that they may bind DNA cooperatively. Indeed, not only did MHF enhance the DNA binding of FANCM754, FANCM754 also stimulated the DNA binding activity of MHF (; the unbound DNA was reduced in lane 6 compared to lane 5). Moreover, FANCM754 and MHF bound DNA synergistically at low protein concentrations: while either protein alone showed little binding activity, both proteins together displayed an activity much higher than the sum of individuals (; less than 1% of dsDNA was shifted in lanes 10-13, whereas 50-90% of DNA was shifted in lanes 14-16). This synergy was also observed for fork and Holliday junction (HJ) substrates (, lanes 17 to 32).
We reconstituted the FANCM-MHF complex by co-expressing full-length recombinant FANCM, MHF1 and MHF2 proteins in insect cells, and purified the trimeric complex (). Incubation of FANCM-MHF with synthetic replication forks led to formation of a defined protein-DNA complex whose mobility was reduced compared to that of FANCM-fork complex, suggesting that FANCM, MHF1 and MHF2 bind together to branched DNA molecules (). Moreover, the FANCM-MHF complex had a stronger fork binding activity than FANCM alone (), providing further evidence that FANCM-MHF binds DNA cooperatively.
MHF stimulates replication fork reversal activity of FANCM
FANCM exhibits an ATP-dependent replication fork reversal activity, which may stabilize stalled forks and facilitate assembly of DNA damage signaling and repair complexes (Gari et al., 2008a
). We found that recombinant FANCM-MHF had a fork reversal activity stronger than that of FANCM (). Both FANCM and FANCM-MHF catalyzed reversal of a model replication fork into a four-way junction intermediate, and led to the formation of a labeled linear DNA duplex, the end product of complete fork reversal (). At low protein concentrations, however, FANCM-MHF produced higher levels of four-way junction intermediate (about 5 to 7 -fold) and linear regression product (about 2 to 3 -fold) than FANCM alone (). These data establish FANCM-MHF as a DNA remodeling machine, and suggest that MHF is a crucial co-factor for FANCM in both binding and ATP-dependent remodeling of DNA.
MHF is required for stability of FANCM and for activation of the FA pathway
We next examined whether MHF is required for FANCM function in vivo. SiRNA depletion of either MHF1 or MHF2 in HeLa cells reduced the level of FANCM in whole cell lysates () and the chromatin fractions (), suggesting that MHF is required for stability of FANCM and may also be important for chromatin association of FANCM. Depletion of one MHF protein also reduced the protein level of the other (), providing additional evidence that MHF1 and MHF2 are direct interacting partners and depend on each other for stability.
MHF is required for stability of FANCM, activation of the FA pathway and cellular resistance to DNA damaging agents
HeLa cells depleted of either MHF protein displayed reduced levels of monoubiquitinated FANCD2 and FANCI in response to DNA crosslinking drugs, mitomycin C (MMC) and cisplatin (). The cells also exhibited hypersensitivity to these drugs ( and S5A
), and increased chromosomal breaks in response to MMC (Figure S5B
). All these phenotypes are characteristics for cells defective in FA core complex components, indicating that MHF is important for normal functions of the core complex and the FA pathway.
We noticed that HeLa cells depleted of FANCM or MHF were sensitive to methyl methanesulfonate (MMS), a DNA alkylating agent (). This feature has been found in hamster cells mutated of FANCG (Tebbs et al., 2005
), and in yeast FANCM (Banerjee et al., 2008
; Sun et al., 2008
) and MHF mutants (see below), suggesting that some DNA repair functions of FANCM-MHF are conserved in lower eukaryotes.
Cells depleted of MHF also exhibited sensitivity to camptothecin (CPT), a topoisomerase I inhibitor (). This feature was observed in cells depleted of FANCM but not in those lacking other components of the FA core complex (Rosado et al., 2009
; Singh et al., 2009
). The results suggest that FANCM-MHF may have functions independent of the FA core complex.
MHF and FANCM are rapidly recruited to DNA interstrand crosslinks (ICLs) that block replication
Because cells depleted of FANCM or MHF are hypersensitive to drugs that induce ICLs, we examined whether FANCM-MHF localizes at ICLs using two independent techniques. First, we utilized laser-activated psoralen conjugates to generate ICLs within a localized region in nucleus, and then visualize proteins recruited to this region by indirect immunofluorescence (Thazhathveetil et al., 2007
). Both FANCM and MHF1 were recruited to the ICLs within 15 min after photoactivation, in a subgroup of cells in random culture (about 15%) ( and data not shown), while they were not recruited to DNA monoadducts generated by laser-activated angelicin (Thazhathveetil et al., 2007
). Co-staining with a cell cycle marker (NPAT) revealed that the recruitment occurred only in S phase cells (Figure S6A, B, C
, and data not shown). When cells were synchronized in S phase, the recruitment was increased to approximately 50% of the cell population (Figure S6D
). The fact that the recruitment of FANCM and MHF to ICLs occurs only during S phase provides in vivo evidence for FANCM-MHF to remodel replication forks blocked by crosslinked DNA. Notably, the recruitment of FANCM (but not γ-H2AX) was strongly diminished in cells depleted of either MHF1 or MHF2 (). This may be due to reduced stability () and/or impaired targeting of FANCM in the absence of MHF.
MHF is rapidly recruited to ICL sites in S phage cells and required for FANCM recruitment
We also used eChIP, a chromatin-IP based method that detects proteins at a site-specific psoralen ICL on an episomal plasmid transfected into cells ()(Shen et al., 2009
). This plasmid contains the replication origin of Epstein-bar virus (EBV), so that the plasmid without the ICL can undergo replication unidirectionally in EBNA-293 cells that express the Epstein-bar nuclear antigen 1 (EBNA). For the plasmid carrying the ICL (which was positioned 488 bp downstream of the replication origin), the replication fork is stalled by the crosslink, and proteins accumulated at the stalled fork can be detected by ChIP-PCR using a primer set that amplifies a DNA fragment near the crosslink. The same plasmid can be introduced into standard 293 cells that lack EBNA for detection of proteins recruited to ICL under non-replicating conditions. We found that MHF1 was enriched about 5-fold at the ICL when the episomal vector was allowed to replicate in EBNA-293 cells (). However, the enrichment was reduced to less than 2-fold in the 293 cells that do not support vector replication. In comparison, FAAP24 was enriched at the ICL under both replicating and non-replicating conditions (), in agreement with previous findings (Shen et al., 2009
). These data are congruent with the notion that MHF functions at the replication forks blocked by ICLs, most likely in the form of the FANCM-MHF complex.
MHF and FANCM act in the same pathway for FANCD2 monoubiquitination and suppression of sister-chromatid exchange (SCE)
We have screened FA patients for mutations in MHF1 and MHF2, but have failed to identify such individuals (data not shown). To study the functions of MHF genetically, we inactivated MHF1
in chicken DT40 cells (Figure S7A, B and C
). Compared to wildtype cells, MHF1-/-
cells exhibited a lower level of FANCM and MHF2, a reduced level of monoubiquitinated FANCD2 (, lanes 1-2), and a decreased number of FANCD2 nuclear foci (Figure S7D and E
). Introduction of human MHF1 into MHF1-/-
cells restored FANCD2 monoubiquitination, and also resulted in over expression of FANCM (2-fold) and MHF2 (11-fold) compared to wildtype cells (, lanes 1-3). These findings are consistent with siRNA data from HeLa cells that MHF1 is required for normal FANCD2 monoubiquitination and for stability of FANCM and MHF2.
MHF and FANCM act in the same pathway for FANCD2 monoubiquitination and suppression of SCE in chicken DT40 Cells
We generated FANCM-/-/MHF1-/--double mutant cells (data not shown), and found that they contained the levels of monoubiquitinated FANCD2 comparable to that of FANCM-/- cells in the presence of MMC (). These results suggest that MHF and FANCM act in a common pathway to promote efficient monoubiquitination of FANCD2.
DT40 cells inactivated of FANC genes exhibit higher levels of SCEs (Rosado et al., 2009
). The level of SCEs in MHF1-/-
cells was about 3 to 4-fold higher than that of wildtype cells (9.7 vs. 2.5), and this elevated SCE level could be corrected by expression of human MHF1 (). The data suggest that MHF participates in suppression of SCEs in DT40 cells. The SCE level in MHF1-/-
cells is lower than that of FANCM-/-
cells (9.7 vs. 18.3), suggesting that without MHF, FANCM remains partially active in maintaining genome integrity (). The SCE level of FANCM-/-/MHF1-/-
cells is comparable to that of FANCM-/-
cells (17.9 vs. 18.3), indicating that MHF and FANCM act through the same pathway to suppress SCEs.
MHF1-/- DT40 cells lacked cellular sensitivity and chromosomal breakage in response to DNA ICL drugs (data not shown), which are phenotypes of DT40 cells inactivated of FANC genes. These results differ from those of siRNA studies in HeLa cells. Possibly, the balance between DNA repair and cell death pathways may be different between these cells.
The DNA binding activity of MHF is required for normal FANCD2 monoubiquitination and full suppression of SCE
To study whether the DNA binding activity of MHF is required for its function in vivo, we generated three MHF1 point mutants by substituting 2 clusters of conserved positively charged amino acid residues with alanine: mutant A (K73A/R74A), B (R87A/R88A), and AB (K73A/R74A/R87A/R88A) (). Mutagenesis of similar residues in other histone-fold proteins can disrupt protein-DNA interactions (Hori et al., 2008
). We co-expressed these mutants with MHF2 in E.coli
, and found that only mutant A can be co-purified with MHF2 in a stable complex ( and data not shown). Notably, the recombinant complex containing mutant A lacked dsDNA binding activity (), and also failed to recruit FANCM to fork DNA (, compare lanes 4 and 6), indicating that MHF requires its DNA binding activity to recruit FANCM to DNA. Co-IP analyses in HEK293 cells showed that mutant A retained normal association with MHF2 and FANCM (). Importantly, MHF1-/-
DT40 cells stably expressing mutant A had a lower level of monoubiquitinated FANCD2 (, lanes 3-4), and a higher SCE frequency () than cells transfected with wildtype MHF1, suggesting that the DNA binding activity of MHF is required for normal FANCD2 monoubiquitination and full suppression of SCE. Compared to MHF1-/-
null cells, the mutant A-expressing MHF1-/-
cells reproducibly exhibited a higher level of monoubiquitinated FANCD2 and a lower SCE frequency (), indicating that this mutant remains partially functional even though it lacks the ability to bind DNA. This partial function might be due to the ability of mutant A to stabilize FANCM and MHF2, as the latter two proteins were recovered to levels higher than not only those of MHF1-/-
null cells, but also those of wildtype cells (, lanes 1, 2 and 4). The findings that mutant A-complemented cells had a higher than normal amount of FANCM but still exhibited abnormal FANCD2 monoubiquitination and SCE frequency suggest that the stabilization and over expression of FANCM cannot substitute the function of MHF in vivo.
The interaction between MHF and FANCM is essential for FANCM stability
We also analyzed MHF1 mutant B and AB using the same assays. These mutants cannot be co-purified with MHF2 in a stable complex from E.coli (data not shown), indicating that protein-interactions between MHF1-MHF2 were impaired. Consistent with such an impairment, co-IP in HEK293 cells showed that mutant B was partially defective in association with MHF2 and FANCM, whereas mutant AB was completely deficient (). Notably, the levels of FANCM and MHF2 in the mutant B-complemented MHF1-/- DT40 cells were restored to near those of cells expressing wildtype MHF1 or mutant A (). This is in contrast to mutant AB-complemented MHF1-/- cells, in which the levels of FANCM and MHF2 were not restored and similar to those of MHF1-/- null cells, suggesting that the stability of these two proteins strongly depends on their interactions with MHF1.
The levels of monoubiquitinated FANCD2 and SCE in mutant B-complemented MHF1-/- cells were found to be intermediate between MHF1-/- null cells and those expressing wildtype MHF1 (). In contrast, the levels in the mutant AB-complemented MHF1-/- cells were indistinguishable to those of the MHF1-/- null cells. The degree of defects in FANCD2 monoubiquitination and SCE by the two mutants appears to correlate with their ability to stabilize FANCM. Together with the data of mutant A, the results suggest that MHF has two important activities: binding to DNA and stabilizing FANCM by protein-protein interactions. Only when both activities are inactivated (as in mutant AB), MHF completely loses its ability to promote FANCD2 monoubiquitination and suppress SCEs.
Budding yeast MHF and FANCM orthologs work in the same pathway in resistance to MMS-induced DNA damage
We investigated whether yeast orthologs of MHF (Figure S2C and D
) and FANCM work together in DNA repair like their vertebrate counterparts. We disrupted budding yeast orthologs of MHF1 and MHF2 in a srs2
mutant background, and found that both mutants displayed hypersensitivity to MMS (, middle panels, compare mhfΔ srs2Δ
doubles with srs2Δ
single mutant). This feature resembles that of a mph1Δ
mutant, which was previously shown to have MMS hypersensitivity in combination with a srs2Δ
mutant (Banerjee et al., 2008
) (). The mhf1Δ mph1Δ
or mhf2Δ mph1Δ
double mutants displayed MMS hypersensitivity similar to that of the mph1Δ
single mutant in the srs2Δ
background (, bottom panels). Furthermore, the survival frequencies of the triple mutant strains at multiple MMS concentrations were statistically indistinguishable from srs2Δ mph1Δ
(). Collectively, these results indicate that both MHF proteins are epistatic with Mph1 in tolerating MMS-induced DNA damage. In addition, a mhf1Δmhf2Δ
double mutant exhibited MMS hypersensitivity indistinguishable from each mhfΔ
single mutant in the srs2Δ
background (, middle panels), consistent with the evidence in mammalian cells that the two proteins work in the same complex. The MMS resistance of the mhf1Δ
mutant is weaker than that of the mph1Δ
mutant, reminiscent of results in DT40 cells where the SCE level of the MHF1-/-
mutant is lower than that of the FANCM-/-
mutant (). FANCM or MPH1 may therefore be partially functional without MHF. Taken together, these data support the findings in mammalian cells and indicate that the MHF proteins play an evolutionarily conserved role--cooperating with MPH1/FANCM to protect genome integrity.
Yeast MHF and FANCM orthologs work in the same pathway to resist MMS-induced DNA damage and promote gene conversion at stalled replication forks
MHF2 and FANCM act in the same pathway to promote gene conversion at stalled replication forks in fission yeast
The FANCM ortholog in S. pombe
, Fml1, promotes Rad51-dependent gene conversion at blocked replication forks (Sun et al., 2008
). To investigate if MHF is also required, we deleted the mhf2
gene and assessed what impact this had on the frequency of Ade+
recombinants induced by a polar replication fork barrier (RTS1
) positioned between a direct repeat of ade6-
heteroalleles on chromosome 3 of S. pombe
(). This region is replicated unidirectionally, and consequently only orientation 2 of RTS1
blocks replication between the repeats. The block of replication results in a strong induction of Ade+
recombinants, which arise both from Rad51-dependent gene conversion, and Rad51-dependent and independent deletions (Sun et al., 2008
). A His3+
gene positioned between the repeats enables these two types of recombinants to be distinguished: Ade+
are conversion-types, whereas Ade+
are deletion-types. In a mhf2Δ
mutant, the frequency of conversion-types in the absence of replication fork blockage at RTS1
(i.e. when RTS1
is in orientation 1) is not significantly different from wild-type (data not shown). However, when RTS1
is in orientation 2 and replication forks are blocked, the frequency of conversion-types is reduced approximately two-fold in a mhf2Δ
mutant compared to wild-type (, left panel). This reduction is statistically significant (P
= <0.0001), but is less than with the fml1Δ
mutant, which shows a 7-fold reduction. Possibly, Fml1 retains some ability to promote gene conversion without MHF. Importantly the frequency of conversion-types in a fml1Δ mhf2Δ
double mutant is the same as in a fml1Δ
single mutant, indicating that Mhf2 acts in the same pathway to promote gene conversion at blocked replication forks as Fml1 does.
Intriguingly, the loss of conversion-types in the mhf2Δ mutant is accompanied by an approximately two-fold increase in deletion-types (, right panel), which again is statistically significant (P = <0.0001). This increase is dependent on Fml1 as it is suppressed in a fml1Δ mhf2Δ double mutant (P = <0.01). These data suggest that MHF may act to prevent Fml1 mediated Rad51-dependent strand invasion events from giving rise to deletions.