Mechanism and substrate specificity of telomeric protein POT1 stimulation of the Werner syndrome helicase

Loss of the RecQ helicase WRN protein causes the cancer-prone progeroid disorder Werner syndrome (WS). WS cells exhibit defects in DNA replication and telomere preservation. The telomeric single-stranded binding protein POT1 stimulates WRN helicase to unwind longer telomeric duplexes that are otherwise poorly unwound. We reasoned that stimulation might occur by POT1 recruiting and retaining WRN on telomeric substrates during unwinding and/or by POT1 loading on partially unwound ssDNA strands to prevent strand re-annealing. To test these possibilities, we used substrates with POT1-binding sequences in the single-stranded tail, duplex or both. POT1 binding to ssDNA tails did not alter WRN activity on nontelomeric duplexes or recruit WRN to telomeric ssDNA. However, POT1 bound tails inhibited WRN activity on telomeric duplexes with a single 3′-ssDNA tail, which mimic telomeric ends in the open conformation. In contrast, POT1 bound tails stimulated WRN unwinding of forked telomeric duplexes. This indicates that POT1 interaction with the ssDNA/dsDNA junction regulates WRN activity. Furthermore, POT1 did not enhance retention of WRN on telomeric forks during unwinding. Collectively, these data suggest POT1 promotes the apparent processivity of WRN helicase by maintaining partially unwound strands in a melted state, rather than preventing WRN dissociation from the substrate

The Werner Syndrome Helicase/Exonuclease Processes Mobile D-Loops through Branch Migration and Degradation

RecQ DNA helicases are critical for preserving genome integrity. Of the five RecQ family members identified in humans, only the Werner syndrome protein (WRN) possesses exonuclease activity. Loss of WRN causes the progeroid disorder Werner syndrome which is marked by cancer predisposition. Cellular evidence indicates that WRN disrupts potentially deleterious intermediates in homologous recombination (HR) that arise in genomic and telomeric regions during DNA replication and repair. Precisely how the WRN biochemical activities process these structures is unknown, especially since the DNA unwinding activity is poorly processive. We generated biologically relevant mobile D-loops which mimic the initial DNA strand invasion step in HR to investigate whether WRN biochemical activities can disrupt this joint molecule. We show that WRN helicase alone can promote branch migration through an 84 base pair duplex region to completely displace the invading strand from the D-loop. However, substrate processing is altered in the presence of the WRN exonuclease activity which degrades the invading strand both prior to and after release from the D-loop. Furthermore, telomeric D-loops are more refractory to disruption by WRN, which has implications for tighter regulation of D-loop processing at telomeres. Finally, we show that WRN can recognize and initiate branch migration from both the 5′ and 3′ ends of the invading strand in the D-loops. These findings led us to propose a novel model for WRN D-loop disruption. Our biochemical results offer an explanation for the cellular studies that indicate both WRN activities function in processing HR intermediates

Correction: Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells.

[This corrects the article DOI: 10.1371/journal.ppat.1009190.]

Inositol phosphates promote HIV-1 assembly and maturation to facilitate viral spread in human CD4+ T cells.

Gag polymerization with viral RNA at the plasma membrane initiates HIV-1 assembly. Assembly processes are inefficient in vitro but are stimulated by inositol (1,3,4,5,6) pentakisphosphate (IP5) and inositol hexakisphosphate (IP6) metabolites. Previous studies have shown that depletion of these inositol phosphate species from HEK293T cells reduced HIV-1 particle production but did not alter the infectivity of the resulting progeny virions. Moreover, HIV-1 substitutions bearing Gag/CA mutations ablating IP6 binding are noninfectious with destabilized viral cores. In this study, we analyzed the effects of cellular depletion of IP5 and IP6 on HIV-1 replication in T cells in which we disrupted the genes encoding the kinases required for IP6 generation, IP5 2-kinase (IPPK) and Inositol Polyphosphate Multikinase (IPMK). Knockout (KO) of IPPK from CEM and MT-4 cells depleted cellular IP6 in both T cell lines, and IPMK disruption reduced the levels of both IP5 and IP6. In the KO lines, HIV-1 spread was delayed relative to parental wild-type (WT) cells and was rescued by complementation. Virus release was decreased in all IPPK or IPMK KO lines relative to WT cells. Infected IPMK KO cells exhibited elevated levels of intracellular Gag protein, indicative of impaired particle assembly. IPMK KO compromised virus production to a greater extent than IPPK KO suggesting that IP5 promotes HIV-1 particle assembly in IPPK KO cells. HIV-1 particles released from infected IPPK or IPMK KO cells were less infectious than those from WT cells. These viruses exhibited partially cleaved Gag proteins, decreased virion-associated p24, and higher frequencies of aberrant particles, indicative of a maturation defect. Our data demonstrate that IP6 enhances the quantity and quality of virions produced from T cells, thereby preventing defects in HIV-1 replication

Viral exploitation of host DNA genome maintenance proteins.

<p>(<b>A</b>) Diagram of a minimal replication protein assembly (replisome) at a viral and a host fork. Topoisomerases, nucleosomes, and chromatin modifiers known to act at both forks are not shown (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Stillman1" target="_blank">[24]</a>). (<b>B</b>) DNA damage signaling in SV40 DNA replication centers at 48 hours post-infection, but not in host DNA replication centers. Mock-infected or SV40-infected BSC40 monkey cells were labeled with 10 µM EdU (a thymidine analog) for 5 minutes to visualize newly replicated DNA. Soluble proteins were pre-extracted and cells were fixed <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Zhao1" target="_blank">[18]</a>. EdU (teal) was coupled to a fluorescent dye using click chemistry (Invitrogen) and DNA was stained with DAPI. Chromatin-bound Tag (green) and histone γH2AX (red) were stained for indirect immunofluorescence as described <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Zhao1" target="_blank">[18]</a>. Cells were visualized with a 63× objective at a 0.6 µm z-axis slice using an Apotome (Zeiss). Scale bars represent 10 µm.</p

Assembly and activation of the SV40 pre-replication complex in vitro.

<p>(<b>A</b>) Domain architecture of SV40 Tag. Three structured domains (yellow) (DnaJ chaperone domain, origin DNA binding domain [OBD], and helicase domain), composed of the zinc (Zn) and AAA+ ATPase sub-domains, are connected by flexible regions (white) (P, cluster of phosphorylated residues that regulates origin activation; HR, host range function). (<b>B</b>) Diagram of ADP-associated SV40 Tag double hexamer bound to the duplex SV40 core origin of DNA replication (EP, central palindrome, AT), with non-origin DNA protruding from the complex (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Cuesta1" target="_blank">[6]</a>). (<b>C</b>) 3D cryo-electron microscopy reveals two conformations (parallel, displaced) of ADP-associated hypo-phosphorylated SV40 Tag double hexamer on SV40 origin DNA as in (B) (adapted from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Cuesta1" target="_blank">[6]</a>). A hypothetical conformation for the activated double hexamer is shown at the right. Dashed lines suggest potential paths of the DNA strands through each protein conformation. (<b>D</b>) Stages of SV40 replication. I, Tag dodecamer assembled on duplex SV40 DNA as in (B); II, hypo-phosphorylated Tag dodecamer activated as in (C) unwinds DNA bidirectionally <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Wessel1" target="_blank">[13]</a> and may assemble host proteins (not shown here) into two sister replisomes that interact physically through the central lobe of the Tag dodecamer; III, hyper-phosphorylation of Tag disrupts interactions between the hexamers <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Fanning2" target="_blank">[7]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Weisshart1" target="_blank">[8]</a>, releasing the replisomes to progress independently along the template chromatin; IV, replication forks converge slowly, accompanied by DNA decatenation, to complete replication, which may involve additional host proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Bullock1" target="_blank">[3]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Shi1" target="_blank">[14]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Zhao1" target="_blank">[18]</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002994#ppat.1002994-Rohaly1" target="_blank">[21]</a>.</p