Cell cycle - Transition and termination of DNA replication

Transition and termination of DNA replication

DNA replication begins in the early S phase. Upon unwinding of DNA by Minichromosome maintenance protein complex (MCM complex) with the help of Origin recognition complex (ORC complex) and Replication proteins (RPA), the DNA polymerase alpha/primase is recruited to DNA (see map Start of DNA replication) [1].

DNA polymerase alpha/primase synthesizes RNA/DNA hybrid on the newly unwound DNA at ~ 30 nucleotides per initiation. Then DNA polymerase delta continues synthesis of this fragment. DNA polymerase delta and DNA polymerase epsilon are activated by proliferating cell nuclear antigen (PCNA) with help of the Replication factor C [2]. PCNA is a homotrimer that forms a ring shaped structure. PCNA binds to the DNA polymerase delta and DNA polymerase epsilon and acts as a ?sliding clamp?, preventing the polymerases from falling 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 to open up the PCNA ring and close it around the template DNA [3].

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

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

Thus, the lagging strand is synthesized discontinuously as a series of RNA-DNA hybrid molecules. Maturation of Okazaki fragments involves removal of the RNA primers (and perhaps some DNA) by flap endonuclease1 (FEN1). RNA primers are cleaved by Ribonuclease H1. Maturated Okazaki fragments are connected by DNA ligase I. 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 by topoisomerases. Topoisomerases are grouped into two types, both of which catalyze the cleavage and regulation of the DNA with the formation of an intermediate that is covalently 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 opposite strand [1]. WRN stimulates the ability of TOP I to relax negatively supercoiled DNA and specifically stimulate the religation step of the relaxation reaction [6], [7].

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

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

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

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

References:

1. Diffley JF, Labib K
   The chromosome replication cycle. Journal of cell science 2002 Mar 1;115(Pt 5):869-72
2. Mossi R, Ferrari E, Hubscher U
   DNA ligase I selectively affects DNA synthesis by DNA polymerases delta and epsilon suggesting differential functions in DNA replication and repair. The Journal of biological chemistry 1998 Jun 5;273(23):14322-30
3. Zhang G, Gibbs E, Kelman Z, O'Donnell M, Hurwitz J
   Studies on the interactions between human replication factor C and human proliferating cell nuclear antigen. Proceedings of the National Academy of Sciences of the United States of America 1999 Mar 2;96(5):1869-74
4. Tom S, Henricksen LA, Bambara RA
   Mechanism whereby proliferating cell nuclear antigen stimulates flap endonuclease 1. The Journal of biological chemistry 2000 Apr 7;275(14):10498-505
5. Tom S, Henricksen LA, Park MS, Bambara RA
   DNA ligase I and proliferating cell nuclear antigen form a functional complex. The Journal of biological chemistry 2001 Jul 6;276(27):24817-25
6. Lebel M, Spillare EA, Harris CC, Leder P
   The Werner syndrome gene product co-purifies with the DNA replication complex and interacts with PCNA and topoisomerase I. The Journal of biological chemistry 1999 Dec 31;274(53):37795-9
7. Laine JP, Opresko PL, Indig FE, Harrigan JA, von Kobbe C, Bohr VA
   Werner protein stimulates topoisomerase I DNA relaxation activity. Cancer research 2003 Nov 1;63(21):7136-46
8. Leppard JB, Champoux JJ
   Human DNA topoisomerase I: relaxation, roles, and damage control. Chromosoma 2005 Jul;114(2):75-85
9. Lou Z, Minter-Dykhouse K, Chen J
   BRCA1 participates in DNA decatenation. Nature structural & molecular biology 2005 Jul;12(7):589-93
10. Levin DS, Bai W, Yao N, O'Donnell M, Tomkinson AE
    An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining. Proceedings of the National Academy of Sciences of the United States of America 1997 Nov 25;94(24):12863-8
11. Dehde S, Rohaly G, Schub O, Nasheuer HP, Bohn W, Chemnitz J, Deppert W, Dornreiter I
    Two immunologically distinct human DNA polymerase alpha-primase subpopulations are involved in cellular DNA replication. Molecular and cellular biology 2001 Apr;21(7):2581-93
12. Henneke G, Koundrioukoff S, Hubscher U
    Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation. Oncogene 2003 Jul 10;22(28):4301-13