Proteomic analysis of p16ink4a-binding proteins.

The p16(ink4a) tumor suppressor protein plays a critical role in cell cycle control, tumorogenesis and senescence. The best known activity for p16(ink4a) is the inhibition of the activity of CDK4 and CDK6 kinases, both playing a key role in cell cycle progression. With the aim to study new p16(ink4a) functions we used affinity chromatography and MS techniques to identify new p16(ink4a)-interacting proteins. We generated p16(ink4a) columns by coupling the protein to activated Sepharose 4B. The proteins from MOLT-4 cell line that bind to p16(ink4a) affinity columns were resolved by SDS-PAGE and identified by MS using a MALDI-TOF. Thirty-one p16(ink4a) -interacting proteins were identified and grouped in functional clusters. The identification of two of them, proliferating cell nuclear antigen (PCNA) and minichromosome maintenance protein 6 (MCM6), was confirmed by Western blotting and their in vivo interactions with p16(ink4a) were demonstrated by immunoprecipitation and immunofluorescence studies. Results also revealed that p16(ink4a) interacts directly with the DNA polymerase delta accessory protein PCNA and thereby inhibits the polymerase activity.

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA.

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA-FEN-1 interaction during DNA replication and repair.

The two DNA clamps Rad9/Rad1/Hus1 complex and proliferating cell nuclear antigen differentially regulate flap endonuclease 1 activity.

DNA damage leads to activation of several mechanisms such as DNA repair and cell-cycle checkpoints. It is evident that these different cellular mechanisms have to be finely co-ordinated. Growing evidence suggests that the Rad9/Rad1/Hus1 cell-cycle checkpoint complex (9-1-1 complex), which is recruited to DNA lesion upon DNA damage, plays a major role in DNA repair. This complex has been shown to interact with and stimulate several proteins involved in long-patch base excision repair. On the other hand, the well-characterised DNA clamp-proliferating cell nuclear antigen (PCNA) also interacts with and stimulates several of these factors. In this work, we compared the effects of the 9-1-1 complex and PCNA on flap endonuclease 1 (Fen1). Our data suggest that PCNA and the 9-1-1 complex can independently bind to and activate Fen1. Finally, acetylation of Fen1 by p300-HAT abolished the stimulatory effect of the 9-1-1 complex but not that of PCNA, suggesting a possible mechanism of regulation of this important repair pathway.

The human Rad9/Rad1/Hus1 damage sensor clamp interacts with DNA polymerase beta and increases its DNA substrate utilisation efficiency: implications for DNA repair.

In eukaryotic cells, checkpoints are activated in response to DNA damage. This requires the action of DNA damage sensors such as the Rad family proteins. The three human proteins Rad9, Rad1 and Hus1 form a heterotrimeric complex (called the 9-1-1 complex) that is recruited onto DNA upon damage. DNA damage also triggers the recruitment of DNA repair proteins at the lesion, including specialized DNA polymerases. In this work, we showed that the 9-1-1 complex can physically interact with DNA polymerase beta in vitro. Functional analysis revealed that the 9-1-1 complex had a stimulatory effect on DNA polymerase beta activity. However, the presence of 9-1-1 complex neither affected DNA polymerase lambda, another X family DNA polymerase, nor the two replicative DNA polymerases alpha and delta. DNA polymerase beta stimulation resulted from an increase in its affinity for the primer-template and the interaction with the 9-1-1 complex stimulated deoxyribonucleotides misincorporation by DNA polymerase beta. In addition, the 9-1-1 complex enhanced DNA strand displacement synthesis by DNA polymerase beta on a 1 nt gap DNA substrate. Our data raise the possibility that the 9-1-1 complex might attract DNA polymerase beta to DNA damage sites, thus connecting directly checkpoints and DNA repair.

The Fen1 extrahelical 3'-flap pocket is conserved from archaea to human and regulates DNA substrate specificity.

Fen1 is a key enzyme for the maintenance of genetic stability in archaea and eukaryotes and is classified as a tumor suppressor. Very recent structural data obtained from Archaeoglobus fulgidus Fen1 suggest that an extrahelical 3'-flap pocket is responsible for substrate specificity, by binding to the unpaired 3'-flap and by opening and kinking the DNA. Since the extrahelical 3'-flap pocket in archaeal Fen1 contains seven amino acids that are conserved to a great extent in human Fen1, we have mutated the four conserved or all seven amino acids in the human Fen1 extrahelical 3'-flap pocket to alanine. Our data suggest that the human extrahelical 3'-flap pocket mutants have lost substrate specificity to the double-flap DNA. Moreover, loss of high affinity for the unpaired 3'-flap suggests that the extrahelical 3'-flap pocket is essential for recognition and processing of the 'physiological' template. Human PCNA could stimulate the human Fen1 extrahelical 3'-flap pocket mutants but not restore their specificity. Thus the substrate specificity of Fen1 has been functionally conserved over a billion years from archaea to human.

The acetylatable lysines of human Fen1 are important for endo- and exonuclease activities.

Human Fen1 can be acetylated in vivo and in vitro resulting in reduced endonuclease and exonuclease activities in vitro. Acetylation occurs at four lysines located at the C terminus of Fen1, which is important for DNA binding. In this paper we show that Fen1 mutant proteins lacking the lysines at the C terminus have both reduced PCNA independent exonucleolytic and endonucleolytic activities. However, lysines at the C terminus are not required for PCNA stimulation of human Fen1. A double flap substrate was optimal for human Fen1 endonuclease and did not require the C-terminal lysines. Similarly, a one nucleotide 3'-overhang nick substrate was optimal for human Fen1 exonuclease and also did not require the C-terminal lysines. Finally, we found by an electromobility shift assay that human Fen1 had a different mode of binding with a double flap substrate containing a one nucleotide 3'-tail when compared to various other flap substrates. Taken together, our results confirm the double flap substrate as the likely in vivo intermediate for human Fen1 and that the C-terminal lysines are important for the endonuclease and exonuclease activities likely through DNA binding.