DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication.

DNA polymerase (pol) ⑀ is thought to be the leading strand replicase in eukaryotes, whereas pols and ␤ are believed to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ⑀ is halted by an abasic site (apurinic/ apyrimidinic (AP) site). In this study, we present in vitro evidence that human pols , ␤, and can perform translesion synthesis (TLS) of an AP site in the presence of pol ⑀, likely by initiating the 3OHs created at the lesion by the arrested pol ⑀. However, in the case of pols and ␤, this TLS requires the presence of a DNA gap downstream from the product synthesized by the pol ⑀, and the optimal gap for efficient TLS is different for the two polymerases. The presence of gaps did not affect the TLS capacity of human pol . Characterization of the reaction products showed that pol ␤ inserted dAMP opposite the AP site, whereas gap filling synthesis by pol resulted in single or double deletions opposite the lesion. The synthesis up to the AP site by pol ⑀ and the subsequent TLS by pols and ␤ are not influenced by human processivity factor proliferating cell nuclear antigen and human single-stranded DNA-binding protein replication protein A. The bypass capacity of pol at the AP site is greatly reduced when a truncated form of the enzyme, which has lost the BRCA1 C-terminal and proline-rich domains, is used. Collectively, our in vitro results support the existence of a mechanism of gap-directed TLS at an AP site involving a switch between the replicative pol ⑀ and the repair pols and ␤.Chromosomal DNA replication in eukaryotic cells requires three DNA polymerases (pols) 3 : pol ␣, pol ␦, and pol ⑀. pol ␣ is the only polymerase that has an associated activity for synthesis of RNA primers and is able to extend from such primers by synthesizing short stretches of DNA (1, 2). Subsequently, processive DNA synthesis is resumed by pol ␦ and/or pol ⑀. Recent work in yeast supports a model wherein, during normal DNA replication, pol ⑀ is primarily responsible for copying the leading strand, and pol ␦ is primarily responsible for copying the lagging strand (3).Abasic sites (AP sites) arise frequently by spontaneous hydrolysis of purines in DNA, represent a common intermediate of numerous DNA repair systems, and are among the most common endogenous DNA lesions generated during normal cell growth (4, 5).An AP site poses a serious problem to the advancement of a pol because the modified base has lost its coding capacity. Accordingly, its replication requires the intervention of one or more Y family polymerases in a process called translesion synthesis or TLS (for reviews see Refs. 6, 7). Recent publications have shown that among these polymerases human pol was able to insert nucleotides opposite the AP site and extend primers further past the lesion in vitro (8). Moreover, pol showed higher abasic lesion bypass capacity in vivo than pols , , and Rev1 (9). Furthermore, it has also been reported that an AP site could be bypassed in vitro by polymerases of other families such as pol ␣ (10), ...

Budding yeast Rad9, like its orthologs, controls two aspects of the cellular response to DNA double strand breaks (DSBs) – signalling of the DNA damage checkpoint and DNA end resection. Rad9 binds to damaged chromatin via modified nucleosomes independently of the cell cycle phase. Additionally, Rad9 engages in a cell cycle-regulated interaction with Dpb11 and the 9-1-1 clamp, generating a second pathway that recruits Rad9 to DNA damage sites. Binding to Dpb11 depends on specific S/TP phosphorylation sites of Rad9, which are modified by cyclin-dependent kinase (CDK). Here, we show that these sites additionally become phosphorylated upon DNA damage. We define the requirements for DNA damage-induced S/TP phosphorylation of Rad9 and show that it is independent of the cell cycle or CDK activity but requires prior recruitment of Rad9 to damaged chromatin, indicating that it is catalysed by a chromatin-bound kinase. The checkpoint kinases Mec1 and Tel1 are required for Rad9 S/TP phosphorylation, but their influence is likely indirect and involves phosphorylation of Rad9 at S/TQ sites. Notably, DNA damage-induced S/TP phosphorylation triggers Dpb11 binding to Rad9, but the DNA damage-induced Rad9-Dpb11 interaction is dispensable for recruitment to DNA damage sites, indicating that the Rad9-Dpb11 interaction functions beyond Rad9 recruitment.