Before a cell divides it has to duplicate its genome so that two identical copies of its DNA content can be partitioned into daughter cells.
In eukaryotic cells, replication is initiated at thousands of origins on the DNA, each resulting in the assembly of two replisomes that travel away from the initiation site in opposite directions.
Complete and high-fidelity duplication of the genome is essential for faithful transmission of genetic information. When DNA replication goes awry, the result could be cells with mutations, missing or extra genetic material a hallmark of the genomic instability seen in most cancers.
Our laboratory aims to investigate processes involved in eukaryotic replication using a combination of conventional biochemistry and single-molecule imaging tools.
To copy their DNA in preparation for cell division, cells must separate the two strands of the double helix. All cells contain a ring-shaped hexameric DNA helicase, which performs this task. Bacteria use DnaB, whereas eukaryotes use the MCM2-7 complex. In G1, two MCM2-7 hexamers assemble around double-stranded DNA (dsDNA) at each origin of replication in a head-to-head fashion (Figure 1).
This structure, known as the pre-replication complex (preRC), remains idle in the G1 phase. In S phase, MCMs are activated through the action of a number of proteins including Cdc45 and GINS, and multiple kinases to subsequently unwind DNA at the replication fork.
To understand the molecular mechanism by which MCM2-7 functions, we developed a single-molecule assay in which a DNA template immobilised on the surface of a microfluidic flow cell is efficiently replicated in soluble extracts derived from Xenopus laevis eggs (Figure 2).
Using this system, we showed that MCM2-7 complexes that initially load as double hexamers on DNA can physically uncouple and function as single hexamers during replication (Yardimci et al Mol Cell, 2010), in contrast to some models. We also showed that MCM2-7 translocates along the leading strand template to unwind DNA suggesting that the helicase goes through a conformational change during activation and transitions from a dsDNA to a single-stranded DNA (ssDNA) binding mode (Fu et al. Cell, 2011). In the future, we aim to gain an in depth understanding of the MCM2-7 dynamics through real-time visualisation of the helicase at the single-molecule level.
Simian Virus 40 (SV40), a mammalian DNA tumor virus, has served as a robust model system for investigating the mechanism of eukaryotic replication for several decades. The virus encodes its own replicative helicase, Large T-antigen (T-ag), which utilises host cell factors for replication of its genome. Our work indicated that T-ag activated at an origin functions as a single hexamer and translocates along ssDNA, similar to the MCM2-7 complex (Yardimci et al Nature, 2012). Importantly, we also discovered a surprising new property of T-ag. We found that T-ag can efficiently bypass a protein adduct covalently cross-linked to the translocation strand. This remarkable plasticity of T-ag may help the SV40 replisome overcome bulky barriers such as DNA-protein cross-links. Current work is focused on defining the molecular mechanism by which a ring-shaped hexamer can overcome large obstacles during translocation.
Figure 2: Single molecule visualization of eukaryotic replication. λ DNA was stretched and immobilized at both 3’ ends on the streptavidin-functionalized surface of a microfluidic flow cell. Immobilized DNA was exposed to Xenopus egg extracts to initiate MCM2-7-dependent replication.
Finally, a second extract containing digoxigenin modified dUTP was withdrawn into the flow cell to confirm bidirectional replication. Extracts were removed via SDS containing buffer, dsDNA was labeled with SYTOX Orangeand, dig-DUTP was labeled with a fluorescent anti-dig antibody. A stretched λ DNA molecule that underwent replication in extracts (bottom). High intensity SYTOX tract corresponds to a replication bubble. Anti-dig tracts coincide with both ends of the bubble indicating bidirectional replication.