The first signal for initiation of replication involves replication licensing factor (RLF), which 'licenses' replication origins by putting them into an initiation-competent state. The second signal, S-phase promoting factor, induces licensed origins to initiate, and in doing so removes the license. RLF of Xenopus can be separated into two essential components, RLF-M and RLF-B, both of which are required for licensing. RLF-M, a fraction containing members of the minichromosome maintenance family, associates with chromatin prior to replication but is removed during replication. Drosophila MCM2 and MCM4 homologs have been identified (See disc proliferation abnormal for information about both of these). RLF-M's reassociation with chromatin requires passage through mitosis. RLF-M requires RLF-B, an as yet uncharacterized fraction, for binding RLF-M to DNA. Apparently RLF binds to origins of replication, but the basis for this binding has not yet been characterized (Chong, 1996 and references).
The focus of all replication forks is the helicase, which catalyzes the transition from double- to single-stranded DNA. In eukaryotes, the identification of the enzyme that acts at chromosomal replication forks awaits further investigation, but the SV40 T antigen fulfills the helicase function in SV40 replication. The origin of replication is selected and identified in yeast by the origin recognition complex, of which one Drosophila homolog (Orc2) has been identified. Identification of other components of the ORC and unraveling the nature of DNA sequences at the origin are currently very active subjects of research.
The replication process is semidiscontinuous. Proteins comprising the replication machine act in concert to unwind the parental strands and carry out the simultaneous synthesis of the two progeny strands. Both progeny strands are synthesized in the 5' to 3' direction, but since parental DNA strands are antiparallel, two distinct mechanisms of DNA synthesis are required. One of the two progeny strands (the leading strand) is synthesized continuously in the direction of fork movement. The other (the lagging strand) is synthesized discontinuously in the direction opposite to fork movement. Discontinuous DNA synthesis on the lagging-strand templates involves the related synthesis of oligoribonucleotide primers, which are then elongated into short DNA chains (Okazaki fragments). Following their synthesis, Okazaki fragements are processed to remove the RNA primers and joined together to form an interrupted progeny strand (Brush, 1996).
As the replication fork advances, a helix-destabilizing protein is required to maintain the single-stranded DNA structure that serves as template for RNA priming and DNA synthesis. In eukaryotes, Replication protein A performs this function. This phosphoprotein consists of three subunits.
DNA synthesis is initiated by the bifunctional pol-alpha:primase complex, a heterotetrameric phosphoprotein. The primase activity resides in the smallest subunit and is tightly associated with the next largest, which is thought to tether the primase to the catalytic subunit. The remaining subunit has no known catalytic function, but it may contribute to recruitment of pol-alpha:primase to the replication fork. The main function of pol-alpha:primase is to serve as a priming enzyme. The primase catalyzes the synthesis of complementary oligoribonucleotides, which are then extended a short distance by the polymerase activity. The pol-alpha:primase serves exclusively to initiate DNA synthesis on the lagging strand, and dissociation from the DNA provides a primer terminus for assembly of the PCNA/pol-delta complex, which serves to extend the RNA/DNA primers originally synthesized by pol-alpha:primase.
The heterodimeric DNA polymerase-delta is involved in the elongation stage of DNA replication, acting both on the leading and lagging strands. Unlike pol-alpha:primase, the polymerase-delta does not act alone but requires the action of two auxiliary factors, the multisubunit replication factor C (RF-C) which binds to the primer terminus of the lagging strand immediately after RNA/DNA primer synthesis has been completed by pol-alpha:primase, allowing the subsequent assembly of a functional pol-delta complex. Once bound the primer-template junction, RF-C loads PCNA onto the DNA in an energy-dependent process. PCNA then functions as a processivity factor, acting as a clamp to prevent sliding of polymerase in the reverse direction. RNase H1 acts with another enzyme to remove the RNA primer used to initiate the lagging strand, while DNA ligase I joins the nacent DNA fragments to complete the synthesis of the lagging strand.
It is not clear how pol-alpha:primase synthesized DNA is proofread, as this enzyme has no exonuclease activity. the catalytic subunit of polymerase-delta contains a 3' to 5' exonuclease activity, functioning in proofreading, which allows high-fidelity DNA synthesis (Brush, 1996).
Brush, G. S. and Kelly, T. J. (1996). Mechanisms for replicating DNA. In "DNA replication in eukaryotic cells". Ed. M. L. DePamphilis. pp 1-43 Cold Spring Harbor Laboratory Press, Plainview, NY.
Chong, J. P. J., Thömmes, P and Blow, J. J. (1996). The role of MCM/P1 proteins in licensing of DNA replication. Trends in Biochem. Sci. 21: 102-106. Medline abstract: 8882583
date revised: 1 December 2007
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