Genome duplication requires exquisite regulation to ensure accurate transmission of genetic information and to prevent chromosomal abnormalities associated with several diseases, including cancer. Further complicating the genome duplication process is the fact that replication forks, the molecular machines responsible for replication of the genome, must be able to stably navigate a dizzying array of replication impediments and chromatin subtypes that pose structural challenges to replication fork progression and stability. Very little is known, however, about how replication forks are able to facilitate progression through difficult-to-replicate regions of the genome present in all eukaryotic cells.
Purification of replication forks from distinct developmental contexts and identification of components provides a means to define plasticity at the fork, an approach for which Drosophila is ideal. In early Drosophila embryos, S phase is three to four minutes in length. Following the maternal-to-zygotic transition, S phase lengthens to approximately one hour. These rapid embryonic S phases are in contrast to the 10-12 hour S phase length in differentiated cell types. Importantly, rapid embryonic S phases of the Drosophila embryo are driven by increased numbers of replication forks per cell. We have developed an iPOND approach, coupled to quantitative mass spectrometry, to enrich and characterize replication forks from Drosophila embryos. This methodology has identified factors that could be important for controlling replication fork progression and activity in development.
One example of how replication fork composition and activity can be modulated during development is the Suppressor of Underreplication protein, SUUR1. In Drosophila polyploid cells, DNA replication is repressed within specific regions of the genome, which results in chromosome fragility. SUUR mediates repression of DNA replication through direct inhibition of replication fork progression. SUUR is not always associated with replication forks, rather it is recruited to active replication forks in specific genomic regions to inhibit their progression. Given that SUUR associates with replication forks in specific developmental contexts, it provides an example of how replication fork composition and activity can be altered during development.
1. Nordman JT, et al. (2014) Cell Rep. 9: 841–9.