Biomedical subjects

Philip J Fay

Publications and source records attributed to Philip J Fay.

26 records · Page 2Linked to original sources

PubMed2003

Template dimerization promotes an acceptor invasion-induced transfer mechanism during human immunodeficiency virus type 1 minus-strand synthesis.

The biochemical mechanism of template switching by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the role of template dimerization were examined. Homologous donor-acceptor template pairs derived from the HIV-1 untranslated leader region and containing the wild-type and mutant dimerization initiation sequences (DIS) were used to examine the efficiency and distribution of transfers. Inhibiting donor-acceptor interaction was sufficient to reduce transfers in DIS-containing template pairs, indicating that template dimerization, and not the mere presence of the DIS, promotes efficient transfers. Additionally, we show evidence that the overall transfer process spans an extended region of the template and proceeds through a two-step mechanism. Transfer is initiated through an RNase H-facilitated acceptor invasion step, while synthesis continues on the donor template. The invasion then propagates towards the primer terminus by branch migration. Transfer is completed with the translocation of the primer terminus at a site distant from the invasion point. In our system, most invasions initiated before synthesis reached the DIS. However, transfer of the primer terminus predominantly occurred after synthesis through the DIS. The two steps were separated by 60 to 80 nucleotides. Sequence markers revealed the position of primer terminus switch, whereas DNA oligomers designed to block acceptor-cDNA interactions defined sites of invasion. Within the region of homology, certain positions on the template were inherently more favorable for invasion than others. In templates with DIS, the proximity of the acceptor facilitates invasion, thereby enhancing transfer efficiency. Nucleocapsid protein enhanced the overall efficiency of transfers but did not alter the mechanism.

5' Untranslated Regions↗

PubMed2002

Mechanism of minus strand strong stop transfer in HIV-1 reverse transcription.

Retrovirus minus strand strong stop transfer (minus strand transfer) requires reverse transcriptase-associated RNase H, R sequence homology, and viral nucleocapsid protein. The minus strand transfer mechanism in human immunodeficiency virus-1 was examined in vitro with purified protein and substrates. Blocking donor RNA 5'-end cleavage inhibited transfers when template homology was 19 nucleotides (nt) or less. Cleavage of the donor 5'-end occurred prior to formation of transfer products. This suggests that when template homology is short, transfer occurs through a primer terminus switch-initiated mechanism, which requires cleavage of the donor 5' terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5' terminus. Transfer was unaffected when donor 5'-end cleavages were blocked but was reduced when internal cleavages within the donor were restricted. Based on the overall data, we conclude that in human immunodeficiency virus-1, which contains a 97-nt R sequence, minus strand transfer occurs through an acceptor invasion-initiated mechanism. Transfer is initiated at internal regions of the homologous R sequence without requiring cleavage at the donor 5'-end. The acceptor invades at gaps created by reverse transcriptase-RNase H in the donor-cDNA hybrid. The fragmented donor is eventually strand-displaced by the acceptor, completing the transfer.

Base Sequence↗

PubMed2002

Altered interactions between the A1 and A2 subunits of factor VIIIa following cleavage of A1 subunit by factor Xa.

Factor VIIIa consists of subunits designated A1, A2, and A3-C1-C2. The limited cofactor activity observed with the isolated A2 subunit is markedly enhanced by the A1 subunit. A truncated A1 (A1(336)) was previously shown to possess similar affinity for A2 and retain approximately 60% of its A2 stimulatory activity. We now identify a second site in A1 at Lys(36) that is cleaved by factor Xa. A1 truncated at both cleavage sites (A1(37-336)) showed little if any affinity for A2 (K(d)>2 microm), whereas factor VIIIa reconstituted with A2 plus A1(37-336)/A3-C1-C2 dimer demonstrated significant cofactor activity ( approximately 30% that of factor VIIIa reconstituted with native A1) in a factor Xa generation assay. These affinity values were consistent with values obtained by fluorescence energy transfer using acrylodan-labeled A2 and fluorescein-labeled A1. In contrast, factor VIIIa reconstituted with A1(37-336) showed little activity in a one-stage clotting assay. This resulted in part from a 5-fold increase in K(m) for factor X when A1 was cleaved at Arg(336). These findings suggest that both A1 termini are necessary for functional interaction of A1 with A2. Furthermore, the C terminus of A1 contributes to the K(m) for factor X binding to factor Xase, and this parameter is critical for activity assessed in plasma-based assays.

Energy Transfer↗

PubMed2002

Strand transfer occurs in retroviruses by a pause-initiated two-step mechanism.

Recombination promotes retrovirus evolution. It involves transferring a growing DNA primer from one genomic RNA template in the virus to the other. Strand transfer results in vitro suggested that pausing of the reverse transcriptase during synthesis allows enhanced RNase H cleavage of the initial, or donor, RNA template that facilitates primer interaction with the acceptor template. Hairpins are common structures in retrovirus RNAs that induce pausing. Analyzing primer transfers in hairpins by base substitution markers showed transfer sites well beyond the site of pausing. We developed methods to distinguish the initial site of primer-acceptor template interaction from the site of primer terminus transfer. The strand transfer mechanism was confirmed to involve two steps. In the first, the acceptor template invades the primer-donor complex. However, the primer terminus continues elongation on the donor RNA. The interacting primer and acceptor strands then propagate by branch migration to catch the advancing primer terminus. Some distance downstream of the invasion site the primer terminus transfers, marking the genetic shift from donor to acceptor. Nucleocapsid protein (NC) is known to influence primer elongation and strand exchange. The presence of NC increased the efficiency of transfers but did not appear to alter the fundamental transfer mechanism.

Base Sequence↗

PubMed2002

Mutations associated with hemophilia A in the 558-565 loop of the factor VIIIa A2 subunit alter the catalytic activity of the factor Xase complex.

The 558-565 loop region in the A2 subunit of factor (F) VIIIa forms a direct interface with FIXa. We have expressed and purified B-domainless FVIII (FVIII(WT)) and B-domainless FVIII containing the hemophilia A-associated mutations Ser558Phe, Val559Ala, Asp560Ala, Gln565Arg, and the activated protein C cleavage site mutant Arg562Ala. Titration of FVIIIa in FXa generation assays showed that the mutant and wild-type proteins had similar functional affinities for FIXa (dissociation constant [K(d)] values approximately 5 nM-20 nM and approximately 100 nM-250 nM in the presence and absence of phospholipid, respectively). The catalytic activities of the factor Xase complex composed of the hemophilia A-associated FVIII species were markedly reduced both in the presence and absence of phospholipid. FVIII(WT) and FVIII(Arg562Ala) showed catalytic rate constant (k(cat)) values of approximately 60 minute(-1) in the presence of phospholipid, whereas the hemophilia A-associated mutants showed k(cat) values ranging from 3.3 minute(-1) to 7.5 minute(-1). In the absence of phospholipid, all k(cat) values were reduced but FVIII(WT) and FVIII(Arg562Ala) retained higher activities as compared with the hemophilic mutant FVIII forms. Fluorescence anisotropy experiments using fluorescein-modified FIXa confirmed that all FVIII forms interacted with FIXa. However, the presence of factor X yielded minimal increases in anisotropy observed with the mutant factor VIII forms, consistent with their reduced activity. These results show that residues within the 558-565 loop are critical in modulating FIXa enzymatic activity but do not contribute significantly to the affinity of FVIIIa for FIXa.

Base Sequence↗

PubMed2002

Ca(2+) binding to both the heavy and light chains of factor VIII is required for cofactor activity.

Previously, we demonstrated that Ca(2+) was necessary for the generation of cofactor activity following reconstitution of factor VIII from its isolated light chain (LC) and heavy chain (HC) but that Ca(2+) did not affect HC-LC binding affinity (Wakabayashi et al. (2001) Biochemistry 40, 10293-10300). Titration of EDTA-treated factor VIII with Ca(2+) followed by factor Xa generation assay showed a two-site binding pattern, with indicated high-affinity (K(d) = 8.9 +/- 1.8 microM) and low-affinity (K(d) = 4.0 +/- 0.6 mM) sites. Analysis by equilibrium dialysis using (45)Ca and <400 microM free Ca(2+) verified a high-affinity binding (K(d) = 18.9 +/- 3.7 microM). Preincubation of either HC or LC with 6 mM Ca(2+) followed by reassociation with the untreated complementary chain in the presence of 0.12 mM Ca(2+) failed to generate significant cofactor activity (<0.5 nM min(-1) (nM LC)(-1)). However, pretreatment of both HC and LC with 6 mM Ca(2+) followed by reassociation (at 0.12 mM Ca(2+)) generated high activity (7.5 +/- 0.4 nM min(-1) (nM LC)(-1)). Progress curves for activity regain following factor VIII-Ca(2+) association kinetics fitted well to a series reaction scheme rather than one of simple association (p < 0.0001), suggesting a multistep process which may include a Ca(2+)-dependent conformational change. These results suggest that factor VIII contains two Ca(2+) binding sites with different affinities and that active factor VIII can be reconstituted from HC and LC only when both chains are preactivated by Ca(2+).

Amino Acid Sequence↗

PubMed2002

Substrate requirements for secondary cleavage by HIV-1 reverse transcriptase RNase H.

During and after minus-strand DNA synthesis, human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) degrades the RNA genome. To remove RNA left after polymerization, the RT aligns to cut 18 nucleotides in from the 5' RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5' end. Transfer of the minus strong stop DNA during viral replication requires cleavage of template RNA. Removal of the terminal RNA segment is a special case because the RNA-DNA hybrid forms a blunt end, shown previously to resist cleavage when tested in vitro. We show here that the structure of the substrate extending beyond the RNA 5' end is an important determinant of cleavage efficiency. A short single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5' end of the viral RNA did not improve the efficiency of secondary cleavage. However, NC protein greatly facilitated the secondary cut on the blunt-ended substrate, suggesting that NC compensates for the unfavorable substrate structure.

Base Sequence↗

PubMed2002

Cofactor activities of factor VIIIa and A2 subunit following cleavage of A1 subunit at Arg336.

Factor VIIIa consists of three subunits designated A1, A2, and A3-C1-C2. The isolated A2 subunit possesses limited cofactor activity in stimulating factor IXa-catalyzed activation of factor X. This activity is markedly enhanced by the A1 subunit (inter-subunit K(d) = 1.8 microm). The C-terminal region of A1 subunit (residues 337-372) is thought to represent an A2-interactive site. This region appears critical to factor VIIIa, because proteolysis at Arg(336) by activated protein C or factor IXa is inactivating. A truncated A1 (A1(336)) showed similar affinity for A2 subunit (K(d) = 0.9 microm) and stimulated its cofactor activity to approximately 50% that observed for native A1. However, A1(336) was unable to reconstitute factor VIIIa activity in the presence of A2 and A3-C1-C2 subunits. Fluorescence anisotropy of fluorescein (Fl)-FFR-factor IXa was differentially altered by factor VIIIa trimers containing either A1 or A1(336). Fluorescence energy transfer demonstrated that, although Fl-A1(336)/A3-C1-C2 bound acrylodan-A2 with similar affinity as the native dimer, an increased inter-fluorophore separation was observed. These results indicate that the C-terminal region of A1 appears necessary to properly orient A2 subunit relative to factor IXa in the cofactor rather than directly stimulate A2 and elucidate the mechanism for cofactor inactivation following cleavage at this site.

2-Naphthylamine↗