DNA unwinding by Mcm4/6/7 is inhibited by the GC-rich sequences on the duplex segment
Our results indicate that thymine-rich single-stranded DNA is required for initial loading and activation of the Mcm helicase. However, it is not known whether thymine sequences are required for processive unwinding of duplex DNA. Therefore, we have examined whether the nucleotide composition of the duplex region affects its unwinding activity. To address this issue, we constructed a series of T-tailed Y-fork structures (T-fork) containing various sequences in the duplex segment. They carried varied contents of cytosine residues on the 3′-tail strand. In gel shift assays, Mcm4/6/7 bound to these Y-fork substrates with identical affinity (Figure 8A), consistent with the notion that the Mcm4/6/7 binds to single-stranded tails. However, in DNA helicase assays, T-fork/(C:G)49 was hardly displaced by Mcm4/6/7, but was readily displaced by SV40 T-antigen DNA helicase (Figure 8B). When thymine or adenine is inserted as every third nucleotide (repeats of CCT or CCA), the extent of unwinding increased (T-fork/(CCT:GGA)16 and T-fork/(CCA:GGT)16). The efficiency of unwinding is roughly correlated with the content of GC pairs in the duplex segment [T-fork/(CCAA:GGTT)12, T-fork/(CCTT:GGAA)12, T-fork/(CAAA:GTTT)12 and T-fork/(CTTT:GAAA)12; Figure 8B]. It appears that the duplex segment containing <50% GC pairs is efficiently unwound (Figure 8C).
We next examined the effect of GC-rich segments on a partial heteroduplex DNA on a single-stranded circular DNA. We have constructed two sets of 5′-tailed partial heteroduplex DNA substrates containing duplex regions of variable lengths; one on M13mp18 vector and the other on M13mp19 containing a 66 nt long G-rich segment downstream of the hybridizing oligonucleotide. We found that the Mcm4/6/7 helicase can displace duplex DNA up to 350 nt long on its own on the former substrate (Figure 8D, left), consistent with previous results (13). In contrast, displacement was inhibited over the GC-rich segments on the latter substrate (Figure 8D, right panel), although displacement up to 250 nt in length, albeit at a reduced level, was observed at a high concentration of Mcm4/6/7 complex. This result is consistent with that on Y-fork and indicates that GC-rich duplex segment is inhibitory for unwinding by the Mcm helicase. These results may indicate that the presence of thymine–adenine pairs with a certain frequency in the duplex region may facilitate continuous unwinding by Mcm4/6/7. Alternatively, Mcm4/6/7 is not efficient enough to displace thermodynamically stable GC-base-paired segment.
In order to distinguish these two possibilities, we designed a new T-fork substrate containing inosine (I) residue instead of guanosine residue. The thermal stability of the I:C base pair is lower than that of the G:C base pair in duplex DNA due to loss of one hydrogen bond and is even lower than A:T base pair (31). We constructed the T-tailed fork substrates containing the (GCC:CGG)10, (GCC:CIG)10 or (GAA:CTT)10 duplex DNA segment (Figure 8E). Mcm4/6/7 hardly displaced the 31 nt long duplex of GCC:CGG repeats, whereas the T-fork/(GAA:CTT)10 was displaced efficiently, as described above. T-fork/(GCC:CIG)10 was displaced with efficiency much greater than that of T-fork/(GCC:CGG)10, indicating that the thermostability of the duplex segment, not the lack of AT base pair, is responsible for inability of Mcm4/6/7 to displace the duplex.