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Cell cycle Transition and termination of DNA replacation


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Cell cycle Transition and termination of DNA replacation

Transition and termination of DNA replication

DNA replication begins in the early S phase. Upon unwinding of DNA byMinichromosome maintenance protein complex (MCM complex) with the help of Originrecognition complex (ORC complex) and Replication proteins (RPA), the DNA polymerasealpha/primase is recruited to DNA (see map ? Start of DNA replication?) [1].

DNA polymerase alpha/primase synthesizes RNA/DNA hybrid on the newly unwoundDNA at ~ 30 nucleotides per initiation. Then DNA polymerase delta continuessynthesis of this fragment. DNA polymerase delta and DNA polymerase epsilonare activated by proliferating cell nuclear antigen ( PCNA ) with help of theReplication factor C [2]. PCNA is a homotrimer that forms a ringshaped structure. PCNA binds to the DNA polymerase delta and DNApolymerase epsilon and acts as a ?sliding clamp?, preventing the polymerases fromfalling off the DNA. Replication factor C is a member of the AAA+ superfamily proteins; it binds to the 3' end of the primer and uses ATP toopen up the PCNA ring and close it around the template DNA [3].

Because of the anti-parallel nature of DNA, the two parental strands arereplicated by different mechanisms during the progression of the replication fork. Theparental strand, which is 3' to 5' relative to the direction of unwinding, can bereplicated continuously by a DNA polymerase alpha/primase and DNA polymerasedelta synthesizing 5' to 3'. This is known as the ?leading strand? [1].

On the other strand (?lagging strand?), however, replication is trickier because DNApolymerases cannot synthesize DNA in a 3' to 5' direction. To circumvent thisproblem, this strand is replicated discontinuously; as the helicase unwinds DNA,DNA polymerase alpha/primase and DNA polymerase delta (and/or DNApolymerase epsilon ) synthesize short oligonucleotides called Okazaki fragments[1].

Thus, the lagging strand is synthesized discontinuously as a series of RNA-DNA hybridmolecules. Maturation of Okazaki fragments involves removal of the RNA primers (andperhaps some DNA) by flap endonuclease1 ( FEN1 ). RNA primers are cleaved byRibonuclease H1. Maturated Okazaki fragments are connected by DNA ligaseI. Activites of FEN1 [4] and DNA ligase I [5] are stimulated by PCNA.

The topology of a DNA molecule changes as it is unwound during DNA replication bytopoisomerases. Topoisomerases are grouped into two types, both of which catalyze thecleavage and regulation of the DNA with the formation of an intermediate that iscovalently bound to DNA through a phosphotyrosine bond. Topoisomerase I ( TOP1 )is monomeric and pass a single-stranded region of DNA through a break in the oppositestrand [1]. WRN stimulates the ability of TOP I to relaxnegatively supercoiled DNA and specifically stimulate the religation step of therelaxation reaction [6], [7].

Type II topoisomerases ( TOP2 ) are homodimeric or heterotetrameric and pass aregion of double-stranded DNA through a break in a second duplex DNA molecule (inter- orintramolecularly) [8]. It is show, that TOP2 may be regulated bytumor suppressor BRCA1 -dependent ubiquitination [9],

Termination occurs when two opposing replication forks meet and the nascent DNA fromthe two forks is ligated together. Replication machinery elements must be displacedbefore the completion of replication to allow the polymerases to replicate the last bitsof sequence [1].

DNA ligase I inhibits DNA polymerase delta via PCNA [10]. Then, formation of new MCM2/ DNA polymerase alpha/primasecomplexes might be prevented by CDK2/ CyclinA phosphorylation of DNApolymerase alpha/primase in late-S-phase [11]. Subsequently CyclinA activates CDK1 which in turn inhibits by phosphorylation functioningFEN1. Phosphorylation of FEN1 by CDK1/ Cyclin A abrogatesits PCNA binding, thus, preventing stimulation of FEN1 by PCNA[12].

Thus, reduplication DNA is completed in late S phase [1].