Iestyn Whitehouse: Using the Lagging Strand to Study Chromosome Replication

Okazaki fragments and nucleosomes

Figure 3: DNA replication Enlarge Image Figure 3: DNA replication The study of DNA synthesis and chromatin assembly at the replication fork is confounded by the highly dynamic nature of the process. At each fork, approximately 25 nucleosomes are assembled per minute, making it very difficult to study the mechanisms of nucleosome assembly and positioning in vivo. Because of this, factors that assemble nucleosomes and complex chromatin structures are poorly understood.  For example, we have little idea how histone chaperones function at the replication fork or whether specialized chaperones function in distinct regions of the genome. Furthermore, we have no information that describes whether and how repositioning of nascent nucleosomes occurs.

DNA replication at the replisome is inherently asymmetric, with the leading strand synthesized in advance of the lagging strand (Figure 3). Okazaki fragment synthesis on the lagging strand necessitates the repeated production of single-stranded DNA and polymerization in the opposite direction to fork progression. Given the temporal delay in synthesis of the lagging strand and the rapidity of histone deposition behind the replication fork, these two processes may be interlinked. However, primarily because eukaryotic Okazaki fragments have never been quantitatively purified and analyzed, such a link has never been rigorously investigated. Thus, although Okazaki fragments constitute approximately 50 percent of all DNA replication, we understand very little about them.

Okazaki fragments to study DNA replication and chromatin

We have developed the first protocols to purify and analyze Okazaki fragments from eukaryotic cells. We have found that the synthesis of Okazaki fragments is closely linked to the process of nucleosome assembly (Smith and Whitehouse, 2012; Figure  4).

Figure 4: Model for Okazaki fragment processing Enlarge Image Figure 4: Model for Okazaki fragment processing Unligated Okazaki fragments leave lasting marks in the genome that provide a record of DNA replication through a particular region. This record can be read by deep sequencing and allows us to dissect locus-specific roles for factors involved in DNA replication and the propagation of epigenetic information. The ability to analyze Okazaki fragments provides tools for the investigation of a vast array of protein factors that act at the replication fork whose functions are ill defined. In addition to unicellular eukaryotes, we are studying Okazaki fragments from metazoans to test whether general principals are conserved.  Finally, analysis of Okazaki fragments may allow us to detect and investigate novel processes that take advantage of the asymmetry at the replication fork.