DNA Replication

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Exploring the intricacies of DNA replication in eukaryotes, this overview highlights the roles of DDK, Sld3, Sld2, and Dpb11 in replication initiation. It delves into the formation of the replisome, the mechanics of the replication fork, strand synthesis, and the maturation of Okazaki fragments. The text also details the functions of the three main replicative DNA polymerases, Pol α, Pol δ, and Pol ε, in maintaining the fidelity and efficiency of DNA replication.

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The Role of DDK in DNA Replication Initiation

DNA replication, a vital cellular process, commences with a highly regulated initiation stage involving several proteins and complexes. The Dbf4-dependent kinase (DDK), consisting of the catalytic subunit Cdc7 and its regulatory partner Dbf4, is crucial in this process. DDK, which is conserved across eukaryotic species, is indispensable for the commencement of the S phase, the phase in which DNA replication occurs. DDK's primary role is to promote the attachment of Cdc45 to the origins of replication on chromatin. It achieves this by phosphorylating the minichromosome maintenance (Mcm) complex, a key element of the pre-replicative complex (pre-RC) that assembles at replication origins during the G1 phase. Recent high-resolution cryo-electron microscopy (cryoEM) studies have shown how Dbf4 binds to the DNA-associated Mcm2-7 double-hexamer, spanning the interface between hexamers and interacting with specific Mcm subunits. Among the Mcm proteins phosphorylated by DDK, the N-terminal serine/threonine-rich domain (NSD) of Mcm4 is particularly critical. This phosphorylation is a vital step in activating the helicase activity of the Mcm complex, which is necessary for unwinding the DNA at replication origins.

Three-dimensional model of double helix DNA with paired nitrogenous bases, sugars and phosphate groups, neutral background and lighting from top left.

Interactions of Sld3, Sld2, and Dpb11 in DNA Replication

The initiation of DNA replication is a complex process that requires the orchestrated interaction of several proteins, including Sld3, Sld2, and Dpb11. Sld3, in complex with Cdc45, binds to the pre-RC at early replication origins during the G1 phase and to later origins during the S phase, with this binding being contingent upon the Mcm complex. Dpb11, meanwhile, interacts with DNA Polymerase ε and facilitates the recruitment of this polymerase to replication origins. The phosphorylation of Sld3 and Sld2 by cyclin-dependent kinase (CDK) is essential for their interaction with Dpb11, which contains two pairs of BRCA1 C Terminus (BRCT) domains that recognize phosphorylated proteins. These interactions are critical for the CDK-mediated activation of DNA replication in budding yeast. Additionally, Dpb11 is involved with the GINS complex, which plays a role in both the initiation and elongation phases of DNA replication. The assembly of a pre-loading complex (pre-LC) comprising Pol ε, GINS, Sld2, and Dpb11 precedes the association with replication origins and is regulated by the combined actions of CDK and DDK.

The Eukaryotic Replisome and the Elongation Phase of DNA Replication

The pre-replicative complex (pre-RC) designates potential sites for DNA replication initiation but does not immediately trigger DNA unwinding or the recruitment of DNA polymerases. The activation of the pre-RC, as cells enter the S phase, leads to the formation of the replisome, a sophisticated machinery responsible for DNA replication. The replisome coordinates the synthesis of DNA, with DNA polymerase ε and DNA polymerase δ responsible for the leading and lagging strands, respectively. It also includes factors such as Claspin, And1, and the replication factor C (RFC) clamp loader, which regulate the activities of the polymerases and synchronize DNA synthesis with the unwinding of the DNA helix by the Cdc45-Mcm-GINS complex. Topoisomerases are also integral to the replisome, as they alleviate the positive supercoils that form ahead of the replication fork, which could otherwise stall replication progress.

Mechanics of the Replication Fork and Strand Synthesis

The replication fork is the dynamic structure where the parental DNA is separated into single strands to serve as templates for replication. DNA polymerases synthesize DNA exclusively in the 5' to 3' direction, necessitating a system for leading and lagging strand synthesis. The leading strand is synthesized continuously in the direction of replication fork progression, while the lagging strand is synthesized discontinuously as Okazaki fragments. The synthesis of the lagging strand is inherently less efficient due to the requirement for multiple RNA primers and the subsequent processing of Okazaki fragments. This processing involves the removal of RNA primers, gap filling by DNA synthesis, and ligation to join the fragments.

The Process of Okazaki Fragment Maturation and Lagging Strand Completion

The maturation of Okazaki fragments is a critical aspect of lagging strand synthesis. Each fragment begins with an RNA primer, which is later displaced and replaced with DNA. The enzymes RNase H and DNA polymerase α participate in the removal of RNA primers and the filling of the resulting gaps. Flap endonuclease 1 (Fen1) processes short flaps that arise from primer displacement, while the enzyme Dna2, which possesses helicase and nuclease activities, processes longer flaps. The final step in lagging strand synthesis is the sealing of nicks by DNA ligase I, which ensures the continuity and integrity of the newly synthesized DNA strand.

Roles of Replicative DNA Polymerases in Eukaryotic DNA Replication

The accurate and complete replication of the eukaryotic genome is facilitated by the coordinated action of three primary replicative DNA polymerases: Polymerase α (Pol α), Polymerase δ (Pol δ), and Polymerase ε (Pol ε). Pol α initiates DNA synthesis by creating a short RNA-DNA primer. Subsequent polymerase switching is necessary for the continuation of DNA synthesis, with Pol ε extending the leading strand and Pol δ synthesizing the Okazaki fragments on the lagging strand. These polymerases, along with other replication factors, work in concert to maintain the fidelity and efficiency of DNA replication.