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PMC:1140370 / 24661-26014
Annnotations
craft-sa-dev
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).
craft-ca-core-ex-dev
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).
craft-ca-core-dev
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).