Preservation of the coding potential of the genome and highly regulated gene expression over the life span of a human are two fundamental requirements of life. These processes require the action of ...repair enzymes or transcription factors that efficiently recognize specific sites of DNA damage or transcriptional regulation within a restricted time frame of the cell cycle or metabolism. A failure of these systems to act results in accumulated mutations, metabolic dysfunction, and disease. Despite the multifactorial complexity of cellular DNA repair and transcriptional regulation, both processes share a fundamental physical requirement that the proteins must rapidly diffuse to their specific DNA-binding sites that are embedded within the context of a vastly greater number of nonspecific DNA-binding sites. Superimposed on the needle-in-the-haystack problem is the complex nature of the cellular environment, which contains such high concentrations of macromolecules that the time frame for diffusion is expected to be severely extended as compared to dilute solution. Here we critically review the mechanisms for how these proteins solve the needle-in-the-haystack problem and how the effects of cellular macromolecular crowding can enhance facilitated diffusion processes. We restrict the review to human proteins that use stochastic, thermally driven site-recognition mechanisms, and we specifically exclude systems involving energy cofactors or circular DNA clamps. Our scope includes ensemble and single-molecule studies of the past decade or so, with an emphasis on connecting experimental observations to biological function.
Abstract
dUTP is a close structural congener of dTTP and can be readily incorporated into DNA opposite to adenine during DNA replication leading to non-mutagenic dU/A base pairs (‘uracilation’). We ...find that dU/A pairs located within DNA transcriptional templates optimized for either T7 RNA polymerase (T7 RNAP) or human RNA polymerase II (pol II) have inhibitory and mutagenic effects on transcription. The data for T7 RNAP establishes that even a single dU/A pair can inhibit promoter binding and transcription initiation up to 30-fold, and that inhibitory effects on transcription elongation are also possible. Sequencing of the mRNA transcribed from uniformly uracilated DNA templates by T7 RNAP indicated an increased frequency of transversion and insertion mutations compared to all T/A templates. Strong effects of dU/A pairs on cellular transcription activity and fidelity were also observed with RNA pol II using uracil base excision repair (UBER)-deficient human cells. At the highest levels of template uracilation, transcription by RNA pol II was completely blocked. We propose that these effects arise from the decreased thermodynamic stability and increased dynamics of dU/A pairs in DNA. The potential implications of these findings on gene regulation and disease are discussed.
SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) is driven into its activated tetramer form by binding of GTP activator and dNTP activators/substrates. In ...addition, the inactive monomeric and dimeric forms of the enzyme bind to single-stranded (ss) nucleic acids. During DNA replication SAMHD1 can be phosphorylated by CDK1 and CDK2 at its C-terminal threonine 592 (pSAMHD1), localizing the enzyme to stalled replication forks (RFs) to promote their restart. Although phosphorylation has only a small effect on the dNTPase activity and ssDNA binding affinity of SAMHD1, perturbation of the native T592 by phosphorylation decreased the thermal stability of tetrameric SAMHD1 and accelerated tetramer dissociation in the absence and presence of ssDNA (∼15-fold). In addition, we found that ssDNA binds competitively with GTP to the A1 site. A full-length SAMHD1 cryo-EM structure revealed substantial dynamics in the C-terminal domain (which contains T592), which could be modulated by phosphorylation. We propose that T592 phosphorylation increases tetramer dynamics and allows invasion of ssDNA into the A1 site and the previously characterized DNA binding surface at the dimer-dimer interface. These features are consistent with rapid and regiospecific inactivation of pSAMHD1 dNTPase at RFs or other sites of free ssDNA in cells.
A major product of oxidative DNA damage is 8-oxoguanine. In humans, 8-oxoguanine DNA glycosylase (hOGG1) facilitates removal of these lesions, producing an abasic (AP) site in the DNA that is ...subsequently incised by AP-endonuclease 1 (APE1). APE1 stimulates turnover of several glycosylases by accelerating rate-limiting product release. However, there have been conflicting accounts of whether hOGG1 follows a similar mechanism. In pre-steady-state kinetic measurements, we found that addition of APE1 had no effect on the rapid burst phase of 8-oxoguanine excision by hOGG1 but accelerated steady-state turnover (k cat) by ∼10-fold. The stimulation by APE1 required divalent cations, could be detected under multiple-turnover conditions using limiting concentrations of APE1, did not require flanking DNA surrounding the hOGG1 lesion site, and occurred efficiently even when the first 49 residues of APE1’s N-terminus had been deleted. Stimulation by APE1 does not involve relief from product inhibition because thymine DNA glycosylase, an enzyme that binds more tightly to AP sites than hOGG1 does, could not effectively substitute for APE1. A stimulation mechanism involving stable protein–protein interactions between free APE1 and hOGG1, or the DNA-bound forms, was excluded using protein cross-linking assays. The combined results indicate a mechanism whereby dynamic excursions of hOGG1 from the AP site allow APE1 to invade the site and rapidly incise the phosphate backbone. This mechanism, which allows APE1 to access the AP site without forming specific interactions with the glycosylase, is a simple and elegant solution to passing along unstable intermediates in base excision repair.
The energetic nature of the interactions of DNA base excision repair glycosylases with undamaged and damaged DNA and the nuclear environment are expected to significantly impact the time it takes for ...these enzymes to search for damaged DNA bases. In particular, the high concentration of monovalent ions, macromolecule crowding, and densely packed DNA chains in the cell nucleus could alter the search mechanisms of these enzymes as compared to findings in dilute buffers typically used in in vitro experiments. Here we utilize an in vitro system where the concerted effects of monovalent ions, macromolecular crowding, and high concentrations of bulk DNA chains on the activity of two paradigm human DNA glycosylases can be determined. We find that the energetic nature of the observed binding free energies of human 8-oxoguanine DNA glycosylase (hOGG1) and human uracil DNA glycosylase (hUNG) for undamaged DNA are derived from different sources. Although hOGG1 uses primarily nonelectrostatic binding interactions with nonspecific DNA, hUNG uses a salt-dependent electrostatic binding mode. Both enzymes turn to a nonelectrostatic mode in their specific complexes with damaged bases in DNA, which enhances damage site specificity at physiological ion concentrations. Neither enzyme was capable of efficiently locating and removing their respective damaged bases in the combined presence of physiological ions and a bulk DNA chain density approximating that found in the nucleus. However, the addition of an inert crowding agent to mimic macromolecular crowding in the nucleus largely restored their ability to track DNA chains and locate damaged sites. These findings suggest how the concerted action of monovalent ions and crowding could contribute to efficient DNA damage recognition in cells.
The HIV-1 restriction factor sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is a tetrameric protein that catalyzes the hydrolysis of all dNTPs to the deoxynucleoside and ...tripolyphosphate, which effectively depletes the dNTP substrates of HIV reverse transcriptase. Here, we establish that SAMHD1 is activated by GTP binding to guanine-specific activator sites (A1) as well as coactivation by substrate dNTP binding to a distinct set of nonspecific activator sites (A2). Combined activation by GTP and dNTPs results in a long-lived tetrameric form of SAMHD1 that persists for hours, even after activating nucleotides are withdrawn from the solution. These results reveal an ordered model for assembly of SAMHD1 tetramer from its inactive monomer and dimer forms, where GTP binding to the A1 sites generates dimer and dNTP binding to the A2 and catalytic sites generates active tetramer. Thus, cellular regulation of active SAMHD1 is not determined by GTP alone but instead, the levels of all dNTPs and the generation of a persistent tetramer that is not in equilibrium with free activators. The significance of the long-lived activated state is that SAMHD1 can remain active long after dNTP pools have been reduced to a level that would lead to inactivation. This property would be important in resting CD4 ⁺ T cells, where dNTP pools are reduced to nanomolar levels to restrict infection by HIV-1.
The HIV-1 restriction factor SAMHD1 is a tetrameric enzyme activated by guanine nucleotides with dNTP triphosphate hydrolase activity (dNTPase). In addition to this established activity, there have ...been a series of conflicting reports as to whether the enzyme also possesses single-stranded DNA and/or RNA 3'-5' exonuclease activity. SAMHD1 was purified using three chromatography steps, over which the DNase activity was largely separated from the dNTPase activity, but the RNase activity persisted. Surprisingly, we found that catalytic and nucleotide activator site mutants of SAMHD1 with no dNTPase activity retained the exonuclease activities. Thus, the exonuclease activity cannot be associated with any known dNTP binding site. Monomeric SAMHD1 was found to bind preferentially to single-stranded RNA, while the tetrameric form required for dNTPase action bound weakly. ssRNA binding, but not ssDNA, induces higher-order oligomeric states that are distinct from the tetrameric form that binds dNTPs. We conclude that the trace exonuclease activities detected in SAMHD1 preparations arise from persistent contaminants that co-purify with SAMHD1 and not from the HD active site. An in vivo model is suggested where SAMHD1 alternates between the mutually exclusive functions of ssRNA binding and dNTP hydrolysis depending on dNTP pool levels and the presence of viral ssRNA.
A fundamental and shared process in all forms of life is the use of DNA glycosylase enzymes to excise rare damaged bases from genomic DNA. Without such enzymes, the highly ordered primary sequences ...of genes would rapidly deteriorate. Recent structural and biophysical studies are beginning to reveal a fascinating multistep mechanism for damaged base detection that begins with short-range sliding of the glycosylase along the DNA chain in a distinct conformation we call the search complex (SC). Sliding is frequently punctuated by the formation of a transient “interrogation” complex (IC) where the enzyme extrahelically inspects both normal and damaged bases in an exosite pocket that is distant from the active site. When normal bases are presented in the exosite, the IC rapidly collapses back to the SC, while a damaged base will efficiently partition forward into the active site to form the catalytically competent excision complex (EC). Here we review the unique problems associated with enzymatic detection of rare damaged DNA bases in the genome and emphasize how each complex must have specific dynamic properties that are tuned to optimize the rate and efficiency of damage site location.
Non-dividing cells of the myeloid lineage such as monocytes and macrophages are target cells of HIV that have low dNTP pool concentrations and elevated levels of dUTP, which leads to frequent ...incorporation of dUMP opposite to A during reverse transcription ("uracilation"). One factor determining the fate of dUMP in proviral DNA is the host cell uracil base excision repair (UBER) system. Here we explore the relative UBER capacity of monocytes (MC) and monocyte-derived macrophages (MDM) and the fate of integrated uracilated viruses in both cell types to understand the implications of viral dUMP on HIV diversification and infectivity. We find that the kinetics for MC infection is compatible with their lifetime in vivo and their near absence of hUNG2 activity is consistent with the retention of viral dUMP at high levels at least until differentiation into macrophages, where UBER becomes possible. Overexpression of human uracil DNA glycosylase in MDM prior to infection resulted in rapid removal of dUMP from HIV cDNA and near complete depletion of dUMP-containing viral copies. This finding establishes that the low hUNG2 expression level in these cells limits UBER but that hUNG2 is restrictive against uracilated viruses. In contrast, overexpression of hUNG2 after viral integration did not accelerate the excision of uracils, suggesting that they may poorly accessible in the context of chromatin. We found that viral DNA molecules with incorporated dUMP contained unique (+) strand transversion mutations that were not observed when dUMP was absent (G→T, T→A, T→G, A→C). These observations and other considerations suggest that dUMP introduces errors predominantly during (-) strand synthesis when the template is RNA. Overall, the likelihood of producing a functional virus from in vitro infection of MC is about 50-fold and 300-fold reduced as compared to MDM and activated T cells. The results implicate viral dUMP incorporation in MC and MDM as a potential viral diversification and restriction pathway during human HIV infection.
Many enzymes that react with specific sites in DNA have the property of facilitated diffusion, in which the DNA chain is used as a conduit to accelerate site location. Despite the importance of such ...mechanisms in gene regulation and DNA repair, there have been few viable approaches to elucidate the microscopic process of facilitated diffusion. Here we describe a new method in which a small-molecule trap (uracil) is used to clock a DNA repair enzyme as it hops and slides between damaged sites in DNA. The 'molecular clock' provides unprecedented information: the mean length for DNA sliding, the one-dimensional diffusion constant, the maximum hopping radius and the time frame for DNA hopping events. In addition, the data establish that the DNA phosphate backbone is a sufficient requirement for DNA sliding.
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