Unwinding of origin-specific structures by human replication protein A occurs in a two-step process

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• Simian virus 40 (SV40) T antigen induces DNA melting at the origin of replication.
• Pseudo-origin DNA (PO-8) with a single-stranded DNA (ssDNA) bubble is bound and denatured by human replication protein A (hRPA).

Purpose of the Study:

• To elucidate the mechanism of hRPA-mediated denaturation of pseudo-origin DNA.
• To understand hRPA's role in the initiation of SV40 DNA replication.

Main Methods:

• Characterization of binding and denaturation parameters for hRPA and PO-8.
• Kinetic analysis of binding and denaturation reactions.
• Temperature-dependent denaturation studies.
• Comparative analysis of various single-stranded DNA-binding proteins (SSBs).

Main Results:

• hRPA dissociation constant for PO-8 is 7.7 x 10(-7) M, higher than for ssDNA (9.0 x 10(-8) M).
• hRPA binding to PO-8 is approximately 4-fold faster than denaturation.
• Denaturation is stimulated nearly 4-fold by increasing temperature from 4 to 37°C; only a fraction of bound substrates are denatured.
• SSB ability to bind PO-8, not denature it, correlates with supporting T antigen-dependent SV40 origin unwinding.

Conclusions:

• hRPA binds pseudo-origins via contacts with both ssDNA and duplex DNA.
• Denaturation occurs on a subset of bound molecules, indicating binding does not always lead to denaturation.
• hRPA's binding capability to pseudo-origins is crucial for SV40 replication initiation.