Single-molecule and ensemble studies of DNA replication system.

摘要

DNA replication is one the most important and complex systems in biology. In this dissertation, we first started from ensemble experiments, studying the clamp (PCNA) and the clamp loader (RFC) from the mesophilic archaeon Methanosarcina acetivorans, and then moved to the single-molecule level trying to reconstitute the DNA replication machinery protein by protein.;In the first study, by developing a real-time fluorescence assay, we discovered that RFC can assemble a PCNA ring from monomers in solution. A motion-based DNA polymerization assay showed that the PCNA assembled by RFC is functional. This PCNA assembly activity required the ATP-bound conformation of RFC. Our work demonstrated a reverse-chaperoning activity for an AAA+ protein that can act as a template for the assembly of another protein complex.;In the second study, by applying fluorescence resonance energy transfer (FRET) to the archaeal replication, we reconstituted part of replication machine, up to four different protein components, at the single-molecule level. We developed a surface-based assay where the loading of the PCNA to the DNA by the RFC was visualized in real time. We discovered an intermediate step likely due to ATP hydrolysis by RFC before PCNA was released on the DNA. Although PCNA itself was not stable as a trimeric ring, once loaded, PCNA remained stably associated with the DNA for hours, allowing us to investigate the subsequent reactions. We found that PCNA prefers to stay near the primer/template junction but still diffuses on both double and single stranded DNA. This is only the second example of direct observation of protein diffusion on single stranded DNA. Diffusion on the single strand, however, is two orders of magnitude slower than on double stranded DNA, and is prevented by cognate single strand binding protein. By adding the DNA polymerase to the loaded clamp, we could follow DNA synthesis by the polymerase-clamp complex by visualizing the motion of the clamp downstream. Interestingly, PCNA frequently slipped back or paused during synthesis, suggesting that spontaneous diffusion of PCNA or its complex with the polymerase is an integral feature even during polymerization.;In the last chapter and appendices, some additional works regarding new instrument development and statistical algorithms for programming are also included.