Cell cycle-specific UNG2 phosphorylations regulate protein turnover, activity and association with RPA

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells

Phosphorylation of human Fen1 by cyclin-dependent kinase modulates its role in replication fork regulation.

Cyclin-dependent kinase (Cdk) Cdk1-Cyclin A can phosphorylate Flap endonuclease 1 (Fen1), a key-enzyme of the DNA replication machinery, in late S phase. Cdk1-cyclin A forms a complex in vitro and in vivo with Fen1. Furthermore, Fen1 phosphorylation is detected in vivo and depends upon Cdks activity. As a functional consequence of phosphorylation by Cdk1-Cyclin A in vitro, endo- and exonuclease activities of Fen1 are reduced whereas its DNA binding is not affected. Moreover, phosphorylation of Fen1 by Cdk1-Cyclin A abrogates its proliferating cell nuclear antigen (PCNA) binding thus preventing stimulation of Fen1 by PCNA. Concomitantly, human cells expressing the S187A mutant defective for Cdk1-Cyclin A phosphorylation accumulate in S phase consistent with a failure in cell cycle regulation through DNA replication. Our results suggest a novel regulatory role of Cdks onto the end of S phase by targeting directly a key enzyme involved in DNA replication

The replication kinase Cdc7-Dbf4 promotes the interaction of the p150 subunit of chromatin assembly factor 1 with proliferating cell nuclear antigen

The coordination of chromatin assembly with DNA replication, which is essential for genomic stability, requires the combined activation of histone deposition with the firing of replication origins. We report here the direct interaction of chromatin assembly factor 1 (CAF1), a key factor involved in histone deposition, with the replication kinase Cdc7-Dbf4. We isolated a complex containing both the largest subunit of CAF1 (p150) and the Cdc7-Dbf4 kinase specifically in S phase and thus prove the existence of this interaction in vivo. We then show that the Cdc7-Dbf4 kinase efficiently phosphorylates p150. This event induces a change in p150 oligomerization state, which promotes binding to proliferating cell nuclear antigen (PCNA). Conversely, CAF1 recruitment is reduced in a PCNA/DNA loading assay using Cdc7-depleted extracts. Our data define p150 as a new target for this kinase with implications for the coordination between DNA replication and CAF1 functions

Signaling from Mus81-Eme2-Dependent DNA Damage Elicited by Chk1 Deficiency Modulates Replication Fork Speed and Origin Usage

International audienceMammalian cells deficient in ATR or Chk1 display moderate replication fork slowing and increased initiation density, but the underlying mechanisms have remained unclear. We show that exogenous deoxyribonucleosides suppress both replication phenotypes in Chk1-deficient, but not ATR-deficient, cells. Thus, in the absence of exogenous stress, depletion of either protein impacts the replication dynamics through different mechanisms. In addition, Chk1 deficiency, but not ATR deficiency, triggers nuclease-dependent DNA damage. Avoiding damage formation through invalidation of Mus81-Eme2 and Mre11, or preventing damage signaling by turning off the ATM pathway, suppresses the replication phenotypes of Chk1-deficient cells. Damage and resulting DDR activation are therefore the cause, not the consequence, of replication dynamics modulation in these cells. Together, we identify moderate reduction of precursors available for replication as an additional outcome of DDR activation. We propose that resulting fork slowing, and subsequent firing of backup origins, helps replication to proceed along damaged templates

cdc2 Cyclin-Dependent Kinase Binds and Phosphorylates Herpes Simplex Virus 1 U(L)42 DNA Synthesis Processivity Factor

Earlier studies have shown that cdc2 kinase is activated during herpes simplex virus 1 infection and that its activity is enhanced late in infection even though the levels of cyclin A and B are decreased below levels of detection. Furthermore, activation of cdc2 requires the presence of infected cell protein no. 22 and the U(L)13 protein kinase, the same gene products required for optimal expression of a subset of late genes exemplified by U(S)11, U(L)38, and U(L)41. The possibility that the activation of cdc2 and expression of this subset may be connected emerged from the observation that dominant negative cdc2 specifically blocked the expression of U(S)11 protein in cells infected and expressing dominant negative cdc2. Here we report that in the course of searching for a putative cognate partner for cdc2 that may have replaced cyclins A and B, we noted that the DNA polymerase processivity factor encoded by the U(L)42 gene contains a degenerate cyclin box and has been reported to be structurally related to proliferating cell nuclear antigen, which also binds cdk2. Consistent with this finding, we report that (i) U(L)42 is able to physically interact with cdc2 at both the amino-terminal and carboxyl-terminal domains, (ii) the carboxyl-terminal domain of U(L)42 can be phosphorylated by cdc2, (iii) immunoprecipitates obtained with anti U(L)42 antibody contained a roscovitine-sensitive kinase activity, (iv) kinase activity associated with U(L)42 could be immunodepleted by antibody to cdc2, and (v) U(L)42 transfected into cells associates with a nocodazole-enhanced kinase. We conclude that U(L)42 can associate with cdc2 and that the kinase activity has the characteristic traits of cdc2 kinase

Proliferating Cell Nuclear Antigen (PCNA) Interactions in Solution Studied by NMR

PCNA is an essential factor for DNA replication and repair. It forms a ring shaped structure of 86 kDa by the symmetric association of three identical protomers. The ring encircles the DNA and acts as a docking platform for other proteins, most of them containing the PCNA Interaction Protein sequence (PIP-box). We have used NMR to characterize the interactions of PCNA with several other proteins and fragments in solution. The binding of the PIP-box peptide of the cell cycle inhibitor p21 to PCNA is consistent with the crystal structure of the complex. A shorter p21 peptide binds with reduced affinity but retains most of the molecular recognition determinants. However the binding of the corresponding peptide of the tumor suppressor ING1 is extremely weak, indicating that slight deviations from the consensus PIP-box sequence dramatically reduce the affinity for PCNA, in contrast with a proposed less stringent PIP-box sequence requirement. We could not detect any binding between PCNA and the MCL-1 or the CDK2 protein, reported to interact with PCNA in biochemical assays. This suggests that they do not bind directly to PCNA, or they do but very weakly, with additional unidentified factors stabilizing the interactions in the cell. Backbone dynamics measurements show three PCNA regions with high relative flexibility, including the interdomain connector loop (IDCL) and the C-terminus, both of them involved in the interaction with the PIP-box. Our work provides the basis for high resolution studies of direct ligand binding to PCNA in solution.This work was supported by the Spanish Ministry of Economic Affairs and Competitiveness (www.mineco.gob.es)grants CTQ2011-28680 to FJB and BIO2009-13265-CO2-01 to IL, and by the grant BIPEDD-CM (P-BIO-0214-2006) from Comunidad Autónoma de Madrid (www.madrid.org) to RCO. ADB was supported by a Juan de la Cierva contract from the Spanish Ministry of Economic Affairs and Competitiveness