Abstract
(English)
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The main scientific progress in the scope of the TMR grant was the 4 collaborations with the Spadari, van der Vliet, Baldacci and Nasheuer labs and it can be summarized as follows (for publications see below):
1. A direct interaction between PCNA and Cdk2 targets PCNA interacting proteins for phosphorylation. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin dependent protein kinases and their regulatory partner, the Cyclins. We showed a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C-termini. We found that PCNA and Cdk2 form a complex together with Cyclin A. This ternary PCNA-Cdk2-Cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of Replication Factor C as well as DNA Ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA Ligase I and to stimulate phosphorylation of DNA Ligase I. Based on our results we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an ' adaptor ' for Cdk2-Cyclin A to PCNA binding DNA replication proteins (collaboration with the van der Vliet group).
2. The current view of DNA replication in eukaryotes predicts that pol a/primage synthesizes the first RNA/DNA primer on the leading strand. Then, replication factor C binds to the 3'-OH end of the nascent DNA strand and loads PCNA and polO, thereby displacing pol a. Pol a has the unique ability to synthesize de novo a short RNA oligonucleotide and to elongate such an RNA primer by incorporation of deoxynucleotides. It displays a low processivity, resulting in synthesis of short RNA/DNA fragments (30 nt), and, due to the lack of an intrinsic or associated 3'->5' exonuclease activity, Pol a is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol a /primase is a critical step in the initiation of DNA synthesis, but little is known on the role of the DNA replication accessory proteins in its regulation. We found that the single
2
stranded DNA binding protein replication protein A (RP-A) acts as an auxiliary factor for pol a playing a dual role: (i) it stabilizes the pol a/primer complex, thus acting as a DNA polymerase clamp, and (ii) it significantly reduces the misincorporation efficiency by pol a. Based on these results, we present a model in which RP~A is involved in the regulation of the early events of DNA synthesis by acting as a 'fidelity clamp' for pol a (collaboration with the Spadari group).
3. Pol 8, is essential for both leading and lagging strand DNA synthesis during chromosomal replicaton in eukaryotes. Pol 8 has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the pre-existing downstream fragment generating an intermediate flap structure which is the target for the Dna2 and Fen 1 endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA primer, we could measure strand displacement by pol 8 coupled to DNA synthesis. Our results suggested that pol 8 alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol 8, thus increasing the processiviy of both synthesis and strand displacement, whereas replication protein A (RP- A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a 'locked' flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase 8 is modulated by the concerted action ofPCNA, RP-A and Fen 1(collaboration with the Spadari group).
4. The subunit that mediates binding of proliferating cell nuclear antigen (PCNA) to human pol 8 for processive DNA synthesis has not yet been clearly identified. We show here that the putative third subunit, of human pol 8, p66, interacts with PCNA through a canonical PCNA-binding sequence located in its C-terminus. Conversely, p66 interacts with the domain-interconnecting loop of PCNA, a region previo~sly shown to be important for pol 8 activity and for binding of the cell cycle inhibit~r p21 Clpl. In accordance with this, a peptide containing the PCNA-binding domain of p21 Clpl inhibited p66 binding to PCNA as well as the activity of native three-subunit pol 8. Furthermore, pull-down assays showed that polymerase 8 require~ p66 for interaction with PCNA. More importantly, only reconstituted three-subunit pol 8 displayed PCNA-dependent DNA replication that could be inhibited by a peptide containing the PCNA-binding domain of p21ciPl. Direct participation of p66 in PCNA-dependent DNA replication in vivo is demonstrated by co- localization of p66 with PCNA and the catalytic subunit (125 ilia) of pol 8 within DNA replication foci. Finally, in vitro phosphorylation of p66 by cyclin-dependent kinases suggests that p66 activity may be subject to cell cycle dependent regulation. Taken together, these results suggested that p66 is the chief mediator of PCNA-dependent DNA synthesis by pol 8 (collaboration with the Baldacci group).
5. Moreover three reviewes have been initiated. One was published in 2000 (Htibscher/Nasheuer; TillS), one in 2001 (Htibscher/Baldaccci, TillS) and one is in press (Htibscher/Spadari, Annu. Rev. Biochem. 2002).
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