Academic journal article Genetics

DNA Replication Control during Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

Academic journal article Genetics

DNA Replication Control during Drosophila Development: Insights into the Onset of S Phase, Replication Initiation, and Fork Progression

Article excerpt

DNA Replication Overview

Before cell division, the genome must be completely and accurately replicated to maintain the integrity of genetic information across cell generations. DNA replication initiates from thousands of DNA elements within the genome called origins of replication. Origins of replication direct the assembly of a large group of proteins and protein complexes to the site that ultimately allow for DNA unwinding and the establishment of two, bidirectional replication forks. DNA ahead of the fork is progressively unwound, generating singlestranded DNA that serves as a template for the synthesis of new DNA (Bleichert et al. 2017; Parker et al. 2017). Through the molecular study of DNA replication initiation and elongation, it is clear that the mechanisms that regulate origin activity and replication fork progression are diverse and complex, particularly in the context of development. Drosophila has provided powerful developmental systems to study both replication initiation and elongation at the cellular and molecular levels (Nordman and Orr-Weaver 2012). Here, we summarize important insights that the Drosophila system has shed upon the regulation of metazoan DNA replication. We then detail seminal studies that have led to critical understanding of the developmental control of replication origin activation and fork elongation. Finally, we address prevailing questions in DNA replication control and the outlook for the field.

Protein Players at the Origin of Replication

DNA replication initiation requires the sequential recruitment and activation of a large number of replication protein components. Unlike in budding yeast, metazoan origins of replication are not defined by any known consensus sequence (Parker et al. 2017). However, protein factors required to establish the replication initiation complex and the replication fork are highly conserved in eukaryotes (Table 1). Replication initiation first requires that origins of replication are bound by the origin recognition complex (ORC) (composed of the six proteins ORC1-6) in late M and G1 phases of the cell cycle (Figure 1). The replication initiation factor cell division cycle 6 (Cdc6) is then recruited to the origin to form a complex with ORC. ORC and Cdc6 work cooperatively to recruit the initiation factor Cdt1 [Double Parked (DUP) in Drosophila] and the six-membered Minichromosome Maintenance (MCM)2-7 replicative helicase complex. In budding yeast, Cdt1 and MCM2-7 form a stable complex in cell lysates and are recruited to origins of replication together (Tanaka and Diffley 2002; Kawasaki et al. 2006; Remus et al. 2009). In Xenopus extracts, however, Cdt1 and MCM2-7 do not coprecipitate, suggesting that Cdt1 and the MCM2-7 complex may be recruited sequentially to replication origins in metazoans (Maiorano et al. 2000).

Two hexamers of the MCM2-7 complex are loaded onto origin DNA in an inactive state before the onset of S phase. Under the regulation of two kinases, S phase Cyclin-Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK), the MCM2-7 complex is joined by CDC45 and the Go-Ichi-Ni-San (GINS) complex, a four-membered complex composed of Sld5, Psf1, Psf2, and Psf3. Together, the CDC45/MCM2-7/GINS (CMG) complex forms the functional replicative helicase (Bleichert et al. 2017; Parker et al. 2017). As two MCM2-7 hexamers are loaded onto a single origin of replication, two CMG complexes establish the independent, bidirectional replication forks after origin activation (Figure 1).

Hurdles for the Molecular Study of Metazoan DNA Replication

Despite the conservation of the proteins governing initiation of DNA replication in eukaryotes, there are complexities in the control of metazoan DNA replication. At the most fundamental level, it remains to be determined what dictates a replication origin and where ORC will bind in metazoans (Prioleau and MacAlpine 2016). This has limited analysis of the regulation of origin activation. …

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