Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

Probing the Conformational Restraints of DNA Damage Recognition with ß-L-Nucleotides.

The DNA building blocks 2'-deoxynucleotides are enantiomeric, with their natural ß-D-configuration dictated by the sugar moiety. Their synthetic ß-L-enantiomers (ßLdNs) can be used to obtain L-DNA, which, when fully substituted, is resistant to nucleases and is finding use in many biosensing and nanotechnology applications. However, much less is known about the enzymatic recognition and processing of individual ßLdNs embedded in D-DNA. Here, we address the template properties of ßLdNs for several DNA polymerases and the ability of base excision repair enzymes to remove these modifications from DNA. The Klenow fragment was fully blocked by ßLdNs, whereas DNA polymerase κ bypassed them in an error-free manner. Phage RB69 DNA polymerase and DNA polymerase ß treated ßLdNs as non-instructive but the latter enzyme shifted towards error-free incorporation on a gapped DNA substrate. DNA glycosylases and AP endonucleases did not process ßLdNs. DNA glycosylases sensitive to the base opposite their cognate lesions also did not recognize ßLdNs as a correct pairing partner. Nevertheless, when placed in a reporter plasmid, pyrimidine ßLdNs were resistant to repair in human cells, whereas purine ßLdNs appear to be partly repaired. Overall, ßLdNs are unique modifications that are mostly non-instructive but have dual non-instructive/instructive properties in special cases.

Bypass of Abasic Site-Peptide Cross-Links by Human Repair and Translesion DNA Polymerases.

DNA-protein cross-links remain the least-studied type of DNA damage. Recently, their repair was shown to involve proteolysis; however, the fate of the peptide remnant attached to DNA is unclear. Particularly, peptide cross-links could interfere with DNA polymerases. Apurinuic/apyrimidinic (AP) sites, abundant and spontaneously arising DNA lesions, readily form cross-links with proteins. Their degradation products (AP site-peptide cross-links, APPXLs) are non-instructive and should be even more problematic for polymerases. Here, we address the ability of human DNA polymerases involved in DNA repair and translesion synthesis (POLß, POLλ, POLη, POLκ and PrimPOL) to carry out synthesis on templates containing AP sites cross-linked to the N-terminus of a 10-mer peptide (APPXL-I) or to an internal lysine of a 23-mer peptide (APPXL-Y). Generally, APPXLs strongly blocked processive DNA synthesis. The blocking properties of APPXL-I were comparable with those of an AP site, while APPXL-Y constituted a much stronger obstruction. POLη and POLκ demonstrated the highest bypass ability. DNA polymerases mostly used dNTP-stabilized template misalignment to incorporate nucleotides when encountering an APPXL. We conclude that APPXLs are likely highly cytotoxic and mutagenic intermediates of AP site-protein cross-link repair and must be quickly eliminated before replication.

Back-Up Base Excision DNA Repair in Human Cells Deficient in the Major AP Endonuclease, APE1.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions generated both by spontaneous base loss and as intermediates of base excision DNA repair. In human cells, they are normally repaired by an essential AP endonuclease, APE1, encoded by the APEX1 gene. Other enzymes can cleave AP sites by either hydrolysis or ß-elimination in vitro, but it is not clear whether they provide the second line of defense in living cells. Here, we studied AP site repairs in APEX1 knockout derivatives of HEK293FT cells using a reporter system based on transcriptional mutagenesis in the enhanced green fluorescent protein gene. Despite an apparent lack of AP site-processing activity in vitro, the cells efficiently repaired the tetrahydrofuran AP site analog resistant to ß-elimination. This ability persisted even when the second AP endonuclease homolog, APE2, was also knocked out. Moreover, APEX1 null cells were able to repair uracil, a DNA lesion that is removed via the formation of an AP site. If AP site hydrolysis was chemically blocked, the uracil repair required the presence of NTHL1, an enzyme that catalyzes ß-elimination. Our results suggest that human cells possess at least two back-up AP site repair pathways, one of which is NTHL1-dependent.

Correlated Target Search by Vaccinia Virus Uracil-DNA Glycosylase, a DNA Repair Enzyme and a Processivity Factor of Viral Replication Machinery.

The protein encoded by the vaccinia virus D4R gene has base excision repair uracil-DNA N-glycosylase (vvUNG) activity and also acts as a processivity factor in the viral replication complex. The use of a protein unlike PolN/PCNA sliding clamps is a unique feature of orthopoxviral replication, providing an attractive target for drug design. However, the intrinsic processivity of vvUNG has never been estimated, leaving open the question whether it is sufficient to impart processivity to the viral polymerase. Here, we use the correlated cleavage assay to characterize the translocation of vvUNG along DNA between two uracil residues. The salt dependence of the correlated cleavage, together with the similar affinity of vvUNG for damaged and undamaged DNA, support the one-dimensional diffusion mechanism of lesion search. Unlike short gaps, covalent adducts partly block vvUNG translocation. Kinetic experiments show that once a lesion is found it is excised with a probability ~0.76. Varying the distance between two uracils, we use a random walk model to estimate the mean number of steps per association with DNA at ~4200, which is consistent with vvUNG playing a role as a processivity factor. Finally, we show that inhibitors carrying a tetrahydro-2,4,6-trioxopyrimidinylidene moiety can suppress the processivity of vvUNG.

Molecular dynamics approach to identification of new OGG1 cancer-associated somatic variants with impaired activity.

DNA of living cells is always exposed to damaging factors. To counteract the consequences of DNA lesions, cells have evolved several DNA repair systems, among which base excision repair is one of the most important systems. Many currently used antitumor drugs act by damaging DNA, and DNA repair often interferes with chemotherapy and radiotherapy in cancer cells. Tumors are usually extremely genetically heterogeneous, often bearing mutations in DNA repair genes. Thus, knowledge of the functionality of cancer-related variants of proteins involved in DNA damage response and repair is of great interest for personalization of cancer therapy. Although computational methods to predict the variant functionality have attracted much attention, at present, they are mostly based on sequence conservation and make little use of modern capabilities in computational analysis of 3D protein structures. We have used molecular dynamics (MD) to model the structures of 20 clinically observed variants of a DNA repair enzyme, 8-oxoguanine DNA glycosylase. In parallel, we have experimentally characterized the activity, thermostability, and DNA binding in a subset of these mutant proteins. Among the analyzed variants of 8-oxoguanine DNA glycosylase, three (I145M, G202C, and V267M) were significantly functionally impaired and were successfully predicted by MD. Alone or in combination with sequence-based methods, MD may be an important functional prediction tool for cancer-related protein variants of unknown significance.

Miscoding and DNA Polymerase Stalling by Methoxyamine-Adducted Abasic Sites.

Apurinic/apyrimidinic (AP) sites appear in DNA spontaneously and as intermediates of base excision DNA repair. AP sites are noninstructive lesions: they strongly block DNA polymerases, and if bypassed, the nature of the incorporated dNMP is mostly guided by the interactions within the polymerase-DNA active site. Many DNA polymerases follow the "A-rule", preferentially incorporating dAMP opposite to natural AP sites. Methoxyamine (MX), a small molecule, efficiently reacts with the aldehyde moiety of natural AP sites, thereby preventing their cleavage by APEX1, the major human AP endonuclease. MX is currently regarded as a possible sensitizer of cancer cells toward DNA-damaging drugs. To evaluate the mutagenic potential of MX, we have studied the utilization of various dNTPs by five DNA polymerases of different families encountering MX-AP adducts in the template in comparison with the natural aldehydic AP site. The Klenow fragment of Escherichia coli DNA polymerase I strictly followed the A-rule with both natural AP and MX-adducted AP sites. Phage RB69 DNA polymerase, a close relative of human DNA polymerases δ and ε, efficiently incorporated both dAMP and dGMP. DNA polymerase ß mostly incorporated dAMP and dCMP, preferring dCMP opposite to the natural AP site and dAMP opposite to the MX-AP site, while DNA polymerase λ was selective for dGMP, apparently via the primer misalignment mechanism. Finally, translesion DNA polymerase κ also followed the A-rule for MX-AP and additionally incorporated dCMP opposite to a natural AP site. Overall, the MX-AP site, despite structural differences, was similar to the natural AP site in terms of the dNMP misincorporation preference but was bypassed less efficiently by all polymerases except for Pol κ.

Recognition of a Clickable Abasic Site Analog by DNA Polymerases and DNA Repair Enzymes.

Azide-alkyne cycloaddition ("click chemistry") has found wide use in the analysis of molecular interactions in living cells. 5-ethynyl-2-(hydroxymethyl)tetrahydrofuran-3-ol (EAP) is a recently developed apurinic/apyrimidinic (AP) site analog functionalized with an ethynyl moiety, which can be introduced into cells in DNA constructs to perform labeling or cross-linking in situ. However, as a non-natural nucleoside, EAP could be subject to removal by DNA repair and misreading by DNA polymerases. Here, we investigate the interaction of this clickable AP site analog with DNA polymerases and base excision repair enzymes. Similarly to the natural AP site, EAP was non-instructive and followed the "A-rule", directing residual but easily detectable incorporation of dAMP by E. coli DNA polymerase I Klenow fragment, bacteriophage RB69 DNA polymerase and human DNA polymerase ß. On the contrary, EAP was blocking for DNA polymerases κ and λ. EAP was an excellent substrate for the major human AP endonuclease APEX1 and E. coli AP exonucleases Xth and Nfo but was resistant to the AP lyase activity of DNA glycosylases. Overall, our data indicate that EAP, once within a cell, would represent a replication block and would be removed through an AP endonuclease-initiated long-patch base excision repair pathway.

Noncatalytic Domains in DNA Glycosylases.

Many proteins consist of two or more structural domains: separate parts that have a defined structure and function. For example, in enzymes, the catalytic activity is often localized in a core fragment, while other domains or disordered parts of the same protein participate in a number of regulatory processes. This situation is often observed in many DNA glycosylases, the proteins that remove damaged nucleobases thus initiating base excision DNA repair. This review covers the present knowledge about the functions and evolution of such noncatalytic parts in DNA glycosylases, mostly concerned with the human enzymes but also considering some unique members of this group coming from plants and prokaryotes.

A New Class of Uracil-DNA Glycosylase Inhibitors Active against Human and Vaccinia Virus Enzyme.

Uracil-DNA glycosylases are enzymes that excise uracil bases appearing in DNA as a result of cytosine deamination or accidental dUMP incorporation from the dUTP pool. The activity of Family 1 uracil-DNA glycosylase (UNG) activity limits the efficiency of antimetabolite drugs and is essential for virulence in some bacterial and viral infections. Thus, UNG is regarded as a promising target for antitumor, antiviral, antibacterial, and antiprotozoal drugs. Most UNG inhibitors presently developed are based on the uracil base linked to various substituents, yet new pharmacophores are wanted to target a wide range of UNGs. We have conducted virtual screening of a 1,027,767-ligand library and biochemically screened the best hits for the inhibitory activity against human and vaccinia virus UNG enzymes. Although even the best inhibitors had IC50 ≥ 100 µM, they were highly enriched in a common fragment, tetrahydro-2,4,6-trioxopyrimidinylidene (PyO3). In silico, PyO3 preferably docked into the enzyme's active site, and in kinetic experiments, the inhibition was better consistent with the competitive mechanism. The toxicity of two best inhibitors for human cells was independent of the presence of methotrexate, which is consistent with the hypothesis that dUMP in genomic DNA is less toxic for the cell than strand breaks arising from the massive removal of uracil. We conclude that PyO3 may be a novel pharmacophore with the potential for development into UNG-targeting agents.

Protein Engineering of DNA-Dependent Enzymes.

Enzymes are extremely efficient natural catalysts of a variety of chemical reactions. Design of enzymes with new functions and properties has become one of the main goals of modern protein engineering. The field of protein engineering is growing intensively, and different strategies were developed for the creation of enzymes with new properties. While there is plenty of methods and instruments, all modern protein engineering strategies could be divided in two major groups, broadly based on the core ideas of rational design or directed evolution. DNA-dependent proteins present an important target of protein engineering due to their wide use in molecular cloning, bioanalytics, and genetic manipulations. Here we review examples of successful application of biochemical, structural and computational approaches belonging to both protein engineering strategies to create new proteins belonging to three important classes of DNA-dependent enzymes: CRISPR-associated nuclease Cas9, DNA polymerases, and DNA glycosylases. The review contains examples of successfully designed enzymes and discusses the most useful approaches in the engineering of these specific enzyme classes, problems restraining the development of this field, and future directions in the development and application of designed DNA-dependent enzymes.

A non-canonical nucleotide from viral genomes interferes with the oxidative DNA damage repair system.

Oxidative damage is a major source of genomic instability in all organisms with the aerobic metabolism. 8-Oxoguanine (8-oxoG), an abundant oxidized purine, is mutagenic and must be controlled by a dedicated DNA repair system (GO system) that prevents G:C→T:A transversions through an easily formed 8-oxoG:A mispair. In some forms, the GO system is present in nearly all cellular organisms. However, recent studies uncovered many instances of viruses possessing non-canonical nucleotides in their genomes. The features of genome damage and maintenance in such cases of alternative genetic chemistry remain barely explored. In particular, 2,6-diaminopurine (Z nucleotide) completely substitutes for A in the genomes of some bacteriophages, which have evolved pathways for dZTP synthesis and specialized polymerases that prefer dZTP over dATP. Here we address the ability of the GO system enzymes to cope with oxidative DNA damage in the presence of Z in DNA. DNA polymerases of two different structural families (Klenow fragment and RB69 polymerase) were able to incorporate dZMP opposite to 8-oxoG in the template, as well as 8-oxodGMP opposite to Z in the template. Fpg, a 8-oxoguanine-DNA glycosylase that discriminates against 8-oxoG:A mispairs, also did not remove 8-oxoG from 8-oxoG:Z mispairs. However, MutY, a DNA glycosylase that excises A from pairs with 8-oxoG, had a significantly lower activity on Z:8-oxoG mispairs. Similar preferences were observed for Fpg and MutY from different bacterial species (Escherichia coli, Staphylococcus aureus and Lactococcus lactis). Overall, the relaxed control of 8-oxoG in the presence of the Z nucleotide may be a source of additional mutagenesis in the genomes of bacteriophages or bacteria that have survived the viral invasion.

Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides.

Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase ß. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

Stalling of Eukaryotic Translesion DNA Polymerases at DNA-Protein Cross-Links.

DNA-protein cross-links (DPCs) are extremely bulky adducts that interfere with replication. In human cells, they are processed by SPRTN, a protease activated by DNA polymerases stuck at DPCs. We have recently proposed the mechanism of the interaction of DNA polymerases with DPCs, involving a clash of protein surfaces followed by the distortion of the cross-linked protein. Here, we used a model DPC, located in the single-stranded template, the template strand of double-stranded DNA, or the displaced strand, to study the eukaryotic translesion DNA polymerases ζ (POLζ), ι (POLι) and η (POLη). POLι demonstrated poor synthesis on the DPC-containing substrates. POLζ and POLη paused at sites dictated by the footprints of the polymerase and the cross-linked protein. Beyond that, POLζ was able to elongate the primer to the cross-link site when a DPC was in the template. Surprisingly, POLη was not only able to reach the cross-link site but also incorporated 1-2 nucleotides past it, which makes POLη the most efficient DNA polymerase on DPC-containing substrates. However, a DPC in the displaced strand was an insurmountable obstacle for all polymerases, which stalled several nucleotides before the cross-link site. Overall, the behavior of translesion polymerases agrees with the model of protein clash and distortion described above.

Boron-containing nucleosides as tools for boron-neutron capture therapy.

Despite the significant progress in cancer cure, the development of new approaches to cancer therapy is still of great importance since many deadly tumors remain untreatable. Boron neutron capture therapy (BNCT), proposed more than eighty years ago, is still considered a potentially advantageous approach. Irradiation of cells containing 10B isotopes with epithermal neutrons and the consequent decay of boron nuclei releases particles that deposit high energy along a very short path, inflicting heavy damage on the target cells but sparing the neighbouring tissue. Delivery and preferential accumulation of boron in cancer cells are the major obstacles that slow down the clinical use of BNCT. Since DNA damage caused by irradiation is the major reason for cell death, the incorporation of boron-containing nucleotides into the DNA of cancer cells may significantly increase the efficacy of BNCT. In this review, we discuss the current state of knowledge in the synthesis of boron-containing nucleosides and their application for BNCT with a special focus on their possible incorporation into genomic DNA.

Translesion activity of PrimPol on DNA with cisplatin and DNA-protein cross-links.

Human PrimPol belongs to the archaeo-eukaryotic primase superfamily of primases and is involved in de novo DNA synthesis downstream of blocking DNA lesions and non-B DNA structures. PrimPol possesses both DNA/RNA primase and DNA polymerase activities, and also bypasses a number of DNA lesions in vitro. In this work, we have analyzed translesion synthesis activity of PrimPol in vitro on DNA with an 1,2-intrastrand cisplatin cross-link (1,2-GG CisPt CL) or a model DNA-protein cross-link (DpCL). PrimPol was capable of the 1,2-GG CisPt CL bypass in the presence of Mn2+ ions and preferentially incorporated two complementary dCMPs opposite the lesion. Nucleotide incorporation was stimulated by PolDIP2, and yeast Pol ζ efficiently extended from the nucleotides inserted opposite the 1,2-GG CisPt CL in vitro. DpCLs significantly blocked the DNA polymerase activity and strand displacement synthesis of PrimPol. However, PrimPol was able to reach the DpCL site in single strand template DNA in the presence of both Mg2+ and Mn2+ ions despite the presence of the bulky protein obstacle.

Displacement of Slow-Turnover DNA Glycosylases by Molecular Traffic on DNA.

In the base excision repair pathway, the initiating enzymes, DNA glycosylases, remove damaged bases and form long-living complexes with the abasic DNA product, but can be displaced by AP endonucleases. However, many nuclear proteins can move along DNA, either actively (such as DNA or RNA polymerases) or by passive one-dimensional diffusion. In most cases, it is not clear whether this movement is disturbed by other bound proteins or how collisions with moving proteins affect the bound proteins, including DNA glycosylases. We have used a two-substrate system to study the displacement of human OGG1 and NEIL1 DNA glycosylases by DNA polymerases in both elongation and diffusion mode and by D4, a passively diffusing subunit of a viral DNA polymerase. The OGG1-DNA product complex was disrupted by DNA polymerase ß (POLß) in both elongation and diffusion mode, Klenow fragment (KF) in the elongation mode and by D4. NEIL1, which has a shorter half-life on DNA, was displaced more efficiently. Hence, both possibly specific interactions with POLß and nonspecific collisions (KF, D4) can displace DNA glycosylases from DNA. The protein movement along DNA was blocked by very tightly bound Cas9 RNA-targeted nuclease, providing an upper limit on the efficiency of obstacle clearance.

Transient protein-protein complexes in base excision repair.

Transient protein-protein complexes are of great importance for organizing multiple enzymatic reactions into productive reaction pathways. Base excision repair (BER), a process of critical importance for maintaining genome stability against a plethora of DNA-damaging factors, involves several enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases and accessory proteins acting sequentially on the same damaged site in DNA. Rather than being assembled into one stable multisubunit complex, these enzymes pass the repair intermediates between them in a highly coordinated manner. In this review, we discuss the nature and the role of transient complexes arising during BER as deduced from structural and kinetic data. Almost all of the transient complexes are DNA-mediated, although some may also exist in solution and strengthen under specific conditions. The best-studied example, the interactions between DNA glycosylases and AP endonucleases, is discussed in more detail to provide a framework for distinguishing between stable and transient complexes based on the kinetic data. Communicated by Ramaswamy H. Sarma.

Variable termination sites of DNA polymerases encountering a DNA-protein cross-link.

DNA-protein cross-links (DPCs) are important DNA lesions induced by endogenous crosslinking agents such as formaldehyde or acetaldehyde, as well as ionizing radiation, cancer chemotherapeutic drugs, and abortive action of some enzymes. Due to their very bulky nature, they are expected to interfere with DNA and RNA synthesis and DNA repair. DPCs are highly genotoxic and the ability of cells to deal with them is relevant for many chemotherapeutic interventions. However, interactions of DNA polymerases with DPCs have been poorly studied due to the lack of a convenient experimental model. We have used NaBH4-induced trapping of E. coli formamidopyrimidine-DNA glycosylase with DNA to construct model DNA polymerase substrates containing a DPC in single-stranded template, or in the template strand of double-stranded DNA, or in the non-template (displaced) strand of double-stranded DNA. Nine DNA polymerases belonging to families A, B, X, and Y were studied with respect to their behavior upon encountering a DPC: Klenow fragment of E. coli DNA polymerase I, Thermus aquaticus DNA polymerase I, Pyrococcus furiosus DNA polymerase, Sulfolobus solfataricus DNA polymerase IV, human DNA polymerases ß, κ and λ, and DNA polymerases from bacteriophages T4 and RB69. Although none were able to fully bypass DPCs in any context, Family B DNA polymerases (T4, RB69) and Family Y DNA polymerase IV were able to elongate the primer up to the site of the cross-link if a DPC was located in single-stranded template or in the displaced strand. In other cases, DNA synthesis stopped 4-5 nucleotides before the site of the cross-link in single-stranded template or in double-stranded DNA if the polymerases could displace the downstream strand. We suggest that termination of DNA polymerases on a DPC is mostly due to the unrelieved conformational strain experienced by the enzyme when pressing against the cross-linked protein molecule.