Biology subjects

Cisneros, G. A.

Publications and source records attributed to Cisneros, G. A..

9 recordsLinked to original sources

bioRxiv2026

Pol γ possesses separate metal binding sites for polymerase and strand displacement functions

Accurate replication of mitochondrial genome (mtDNA) integrity, which is essential for cellular metabolism and energy supply, relies primarily on DNA polymerase gamma (Pol {gamma}), Twinkle helicase, and mitochondrial single-stranded DNA binding protein (mtSSB). Twinkle alone exhibits little helicase activity while reports indicate that Pol {gamma} displays from modest to limited unwinding activity. This led us to dissect Pol {gamma} strand displacement activity using structural, biochemical and in silico approaches. Here, we show that human Pol {gamma} carries out robust strand displacement synthesis at physiological concentrations of divalent metal ions which reveals that distinct metal-binding sites can independently regulate DNA synthesis and unwinding activities. We further showed that Pol {gamma} can displace RNA/DNA hybrid with comparable efficiency as DNA/DNA duplex, representing a key implication on RNA primer removal to preserve mtDNA integrity. Our cryo-electron microscopy structures of Pol {gamma} complexed with a template containing downstream dsDNA and an incoming nucleotide revealed the structural mechanism for the strand displacement activity. We identified four conformational states that represent successive stages of DNA unwinding, accompanied by coordinated rearrangement of the downstream DNA and Pol {gamma} elements that mediate strand displacement. This work establishes biochemical and structural mechanisms of Pol {gamma} strand displacement activity, providing fundamental insight into human mitochondrial DNA replication and integrity. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/701366v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1be1dborg.highwire.dtl.DTLVardef@8912c5org.highwire.dtl.DTLVardef@12f5e75org.highwire.dtl.DTLVardef@e25d2a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Effects of a Cancer-Associated Mutation and Multiple SerinePhosphorylation on Poly(ADP-Ribose) Polymerase 2

Poly [ADP-ribose] polymerase 2 (PARP2) plays a crucial role in DNA repair. A common single-nucleotide polymorphism (SNP) in the PARP2 gene, rs3093921, has been associated with pancreatic cancer and marginal zone lymphoma (MZL). This SNP results in a missense mutation, D235G, in the PARP2 protein. PARP2 is also reported to undergo post-translational modifications (PTMs), particularly phosphorylation at serine residues 226, 232, and 353. The C-terminal region of PARP2 includes the Trp-Gly-Arg (WGR) and ADP-ribosyl transferase (ART) domains, and the helical subdomain (HD). The latter two, spanning residues 220 to 583, comprise the catalytic region of PARP2. The DNA-induced enzymatic activation of PARP2 is regulated by local destabilization of the HD domain. We used molecular dynamics (MD) simulations to investigate the impact of these three PTMs on both the wild-type (WT) and the mutant (D235G) forms of PARP2. Our simulations suggest that, while neither the cancer-associated mutation nor PTMs on their own significantly alter the overall flexibility of residues within the HD domain, they cause notably greater deviations in the backbone structure of the HD domain compared to the WT. In addition, PTMs in the context of the mutation show reduced interactions between the mutation site and other protein regions, likely due to structural stabilization induced by the PTMs. Importantly, PTMs mitigate the structural disruption caused by the mutation, helping the mutant protein retain a WT-like conformation. Additionally, the HD domain contributes to maintaining PARP2 in its inactive state through its connection with the ART domain. However, the D235G mutation weakens this connection. While PTMs alone do not have a significant impact on this connection, the simultaneous presence of both the mutation and PTMs partially alleviates the disruptive effect of the mutation and leads to partial restoration of the connection between the HD and ART domains.

biophysics↗

bioRxiv2024

Impact of a Cancer-Associated Mutation on Poly(ADP-ribose) Polymerase1 Inhibition

Poly(ADP-ribose) polymerase1 (PARP1) plays a vital role in DNA repair and its inhibition in cancer cells may cause cell apoptosis. In this study, we investigated the effects of a PARP1 variant, V762A, which is strongly associated with several cancers in humans, on the inhibition of PARP1 by three FDA approved inhibitors: niraparib, rucaparib and talazoparib. Our work suggests that these inhibitors bind to the V762A mutant more effectively than to the wild-type (WT), with similar binding free energies between them. Talazoparib inhibition uniquely lowers the average residue fluctuations in the mutant than the WT including lower fluctuations of mutants N- and C-terminal residues, conserved H-Y-E traid residues and donor loop (D-loop) residues which important for catalysis more effectively than other inhibitions. However, talazoparib also enhances destabilizing interactions between the mutation site in the HD domain in the mutant than WT. Further, talazoparib inhibition significantly disrupts the functional fluctuations of terminal regions in the mutant, which are otherwise present in the WT. Lastly, the mutation and inhibition do not significantly affect PARP1s essential dynamics. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=144 SRC="FIGDIR/small/623412v1_fig1a.gif" ALT="Figure 1A"> View larger version (45K): org.highwire.dtl.DTLVardef@4d4562org.highwire.dtl.DTLVardef@10643dborg.highwire.dtl.DTLVardef@511ae2org.highwire.dtl.DTLVardef@bc2403_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure.C_FLOATNO Impact of V762A mutation on inhibition of PARP1. C_FIG

biophysics↗

bioRxiv2024

Structure of human MUTYH and functional profiling of cancer-associated variants reveal an allosteric network between its cluster cofactor and active site required for DNA repair

MUTYH is a clinically important DNA glycosylase that thwarts mutations by initiating base-excision repair at 8-oxoguanine (OG):A lesions. The roles for its [4Fe-4S] cofactor in DNA repair remain enigmatic. Functional profiling of cancer-associated variants near the [4Fe-4S] cofactor revealed that most variations abrogate both retention of the cofactor and enzyme activity. Surprisingly, R241Q and N238S retained the metal center and bound substrate DNA tightly but were completely inactive. We solved the crystal structure of human MUTYH bound to a transition state mimic and this revealed that Arg241 and Asn238 build an H-bond network connecting the [4Fe-4S] cluster to the catalytic Asp236 that mediates base excision. The structure of the bacterial MutY variant R149Q, along with molecular dynamics simulations of the human enzyme, show the cofactor functions to position and activate the catalytic Asp. These results suggest that allosteric cross-talk between the DNA binding [4Fe-4S] cofactor and the base excision site regulate DNA repair.

biochemistry↗

bioRxiv2023

Molecular basis for short-chain thioester hydrolysis by acyl hydrolase domains in trans-acyltransferase polyketide synthases

Polyketide synthases (PKSs) are multi-domain enzymatic assembly lines that biosynthesise a wide selection of bioactive natural products from simple building blocks. In contrast to their cis-acyltransferase (AT) counterparts, trans-AT PKSs rely on stand-alone AT domains to load extender units onto acyl carrier protein (ACP) domains embedded in the core PKS machinery. Trans-AT PKS gene clusters also encode acyl hydrolase (AH) domains, which are predicted to share the overall fold of AT domains, but hydrolyse aberrant acyl chains from ACP domains, thus ensuring efficient polyketide biosynthesis. How such domains specifically target short acyl chains, in particular acetyl groups, tethered as thioesters to the substrate-shuttling ACP domains, with hydrolytic rather than acyl transfer activity, has remained unclear. To answer these questions, we solved the first structure of an AH domain and performed structure-guided activity assays on active site variants. Our results offer key insights into chain length control and selection against coenzyme A-tethered substrates, and clarify how the interaction interface between AH and ACP domains contributes to recognition of cognate and non-cognate ACP domains. Combining our experimental findings with molecular dynamics simulations allowed for the production of a data-driven model of an AH:ACP domain complex. Our results advance the currently incomplete understanding of polyketide biosynthesis by trans-AT PKSs, and provide foundations for future bioengineering efforts.

biochemistry↗

bioRxiv2021

Computational Compensatory Mutation Discovery Approach: Predicting a PARP1 Variant Rescue Mutation

The prediction of protein mutations that affect function may be exploited for multiple uses. In the context of disease variants, the prediction of compensatory mutations that reestablish functional phenotypes could aid in the development of genetic therapies. In this work, we present an integrated approach that combines coevolutionary analysis and molecular dynamics (MD) simulations to discover functional compensatory mutations. This approach is employed to investigate possible rescue mutations of a poly(ADP-ribose) polymerase 1 (PARP1) variant, PARP1 V762A, associated with lung cancer and follicular lymphoma. MD simulations show PARP1 V762A exhibits noticeable changes in structural and dynamical behavior compared with wild type PARP1. Our integrated approach predicts A755E as a possible compensatory mutation based on coevolutionary information, and molecular simulations indicate that the PARP1 A755E/V762A double mutant exhibits similar structural and dynamical behavior to WT PARP1. Our methodology can be broadly applied to a large number of systems where single nucleotide polymorphisms (SNPs) have been identified as connected to disease and can shed light on the biophysical effects of such changes as well as provide a way to discover potential mutants that could restore wild type-like functionality. This can in turn be further utilized in the design of molecular therapeutics that aim to mimic such compensatory effect. Significance StatementDiscovering protein mutations with desired phenotypes can be challenging due to its combinatorial nature. Herein we employ a methodology combining gene SNP association to disease, direct coupling analysis and molecular dynamics simulations to systematically predict rescue mutations. Our workflow identifies A755E as a potential rescue for the PARP1 V762A mutation, which has been associated with cancer. This methodology is general and can be applied broadly.

biophysics↗

bioRxiv2021

Divergence in dimerization and activity of primate APOBEC3C

The APOBEC3 (A3) family of single-stranded DNA cytidine deaminases are host restriction factors that inhibit lentiviruses, such as HIV-1, in the absence of the Vif protein that causes their degradation. Deamination of cytidine in HIV-1 (-)DNA forms uracil that causes inactivating mutations when uracil is used as a template for (+)DNA synthesis. For APOBEC3C (A3C), the chimpanzee and gorilla orthologues are more active than human A3C, and the Old World Monkey A3C from rhesus macaque (rh) is not active against HIV-1. Multiple integrated analyses determined why rhA3C was not active against HIV-1 and how to increase this activity. Biochemical, virological, and coevolutionary analyses combined with molecular dynamics simulations showed that the key amino acids needed to promote rhA3C antiviral activity also promoted dimerization. Although rhA3C shares a similar dimer interface with hominid A3C, the key amino acid contacts were different. Overall, our results determine the basis for why rhA3C is less active than human A3C, establish the amino acid network for dimerization and increased activity, and track the loss and gain of A3C antiviral activity in primates. The coevolutionary analysis of the A3C dimerization interface provides a basis from which to analyze dimerization interfaces of other A3 family members.

biochemistry↗

bioRxiv2020

Induced mutagenesis by the DNA cytosine deaminase APOBEC3H Haplotype I protects against lung cancer

The DNA cytosine deaminases APOBEC3A, APOBEC3B, and APOBEC3H Haplotype I can induce mutations in cells that lead to cancer evolution. The database of cancer biomarkers in DNA repair genes (DNArCdb) identified a single nucleotide polymorphism (rs139298) of APOBEC3H Haplotype I that is involved in lung cancer1. Here, we show that this single nucleotide polymorphism causes the destabilization of APOBEC3H Haplotype I. Computational analysis suggests that the resulting K121E change affects the structure of APOBEC3H leading to active site disruption and destabilization of the RNA-mediated dimer interface. A K117E mutation in a K121E background stabilized the APOBEC3H Haplotype I, enabled biochemical study, and showed that the K121E affected catalytic activity, single-stranded DNA binding, and oligomerization on single-stranded DNA. That the destabilization of a DNA mutator would be associated with lung cancer suggests that too much mutation could result in immune recognition or death of tumor cells, suggesting that multiple APOBEC3s would not be expressed in the same tumor cells. In support of this hypothesis, stably expressed APOBEC3H Haplotype I caused a high amount of double-stranded DNA breaks in a lung cancer cell line that endogenously expressed APOBEC3B. Altogether, the data support the model that high APOBEC3 mutations in tumors are protective.

biophysics↗

bioRxiv2017

Computational Investigation of O2 Diffusion Through an Intra-molecular Tunnel in AlkB; Influence of Polarization on O2 Transport

E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular Oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature, 439, 879) proposed the existence of an intra-molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra-molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co-substrate out of the active site, compared with the non-polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.

biochemistry↗