DNA repair in higher plants; photoreactivation is the major DNA repair pathway in non-proliferating cells while excision repair (nucleotide excision repair and base excision repair) is active in proliferating cells

We investigated expression patterns of DNA repair genes such as the CPD photolyase, UV-DDB1, CSB, PCNA, RPA32 and FEN-1 genes by northern hybridization analysis and in situ hybridization using a higher plant, rice (Oryza sativa L. cv. Nipponbare). We found that all the genes tested were expressed in tissues rich in proliferating cells, but only CPD photolyase was expressed in non-proliferating tissue such as the mature leaves and elongation zone of root. The removal of DNA damage, cyclobutane pyrimidine dimers and (6–4) photoproducts, in both mature leaves and the root apical meristem (RAM) was observed after UV irradiation under light. In the dark, DNA damage in mature leaves was not repaired efficiently, but that in the RAM was removed rapidly. Using a rice 22K custom oligo DNA microarray, we compared global gene expression patterns in the shoot apical meristem (SAM) and mature leaves. Most of the excision repair genes were more strongly expressed in SAM. These results suggested that photoreactivation is the major DNA repair pathway for the major UV-induced damage in non-proliferating cells, while both photoreactivation and excision repair are active in proliferating cells.     

A 32P-postlabeling assay for the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine in mammalian tissues: evidence that four type II I-compounds are dinucleotides containing the lesion in the 3' nucleotide.

8,5'-Cyclopurine-2'-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of (32)P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5'-cyclo-2'-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic (32)P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The (32)P-postlabeling technique used here is capable of detecting 1-5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.     

The type of DNA glycosylase determines the base excision repair pathway in mammalian cells.

The base excision repair (BER) of modified nucleotides is initiated by damage-specific DNA glycosylases. The repair of the resulting apurinic/apyrimidinic site involves the replacement of either a single nucleotide (short patch BER) or of several nucleotides (long patch BER). The mechanism that controls the selection of either BER pathway is unknown. We tested the hypothesis that the type of base damage present on DNA, by determining the specific DNA glycosylase in charge of its excision, drives the repair of the resulting abasic site intermediate to either BER branch. In mammalian cells hypoxanthine (HX) and 1,N6-ethenoadenine (epsilonA) are both substrates for the monofunctional 3-methyladenine DNA glycosylase, the ANPG protein, whereas 7,8-dihydro-8-oxoguanine (8-oxoG) is removed by the bifunctional DNA glycosylase/beta-lyase 8-oxoG-DNA gly- cosylase (OGG1). Circular plasmid molecules containing a single HX, epsilonA, or 8-oxoG were constructed. In vitro repair assays with HeLa cell extracts revealed that HX and epsilonA are repaired via both short and long patch BER, whereas 8-oxoG is repaired mainly via the short patch pathway. The preferential repair of 8-oxoG by short patch BER was confirmed by the low efficiency of repair of this lesion by DNA polymerase beta-deficient mouse cells as compared with their wild-type counterpart. These data fit into a model where the intrinsic properties of the DNA glycosylase that recognizes the lesion selects the branch of BER that will restore the intact DNA template.     

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.     

Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.     

Modeling the interactions of the nucleotide excision repair UvrA(2) dimer with DNA

The UvrA protein initiates the DNA damage recognition process by the bacterial nucleotide excision repair (NER) system. Recently, crystallographic structures of holo-UvrA(2) dimers from two different microorganisms have been released (Protein Data Bank entries 2r6f , 2vf7 , and 2vf8 ). However, the details of the DNA binding by UvrA(2) and other peculiarities involved in the damage recognition process remain unknown. We have undertaken a molecular modeling approach to appraise the possible modes of DNA-UvrA(2) interaction using molecular docking and short-scale guided molecular dynamics [continuum field, constrained, and/or unrestricted simulated annealing (SA)], taking into account the three-dimensional location of a series of mutation-identified UvrA residues implicated in DNA binding. The molecular docking was based on the assumptions that the UvrA(2) dimer is preformed prior to DNA binding and that no major protein conformational rearrangements, except moderate domain reorientations, are required for binding of undamaged DNA. As a first approximation, DNA was treated as a rigid ligand. From the electrostatic relief of the ventral surface of UvrA(2), we initially identified three, noncollinear DNA binding paths. Each of the three resulting nucleoprotein complexes (C1, C2, and C3) was analyzed separately, including calculation of binding energies, the number and type of interaction residues (including mutated ones), and the predominant mode of translational and rotational motion of specific protein domains after SA to ensure improved DNA binding. The UvrA(2) dimer can accommodate DNA in all three orientations, albeit with different binding strengths. One of the UvrA(2)-DNA complexes (C1) fulfilled most of the requirements (high interaction energy, proximity of DNA to mutated residues, etc.) expected for a natural, high-affinity DNA binding site. This nucleoprotein presents a structural organization that is designed to clamp and bend double-stranded DNA. We examined the binding site in more detail by docking DNAs of significantly different (AT- vs CG-enriched) sequences and by submitting the complexes to DNA-unrestricted SA. It was found that in a manner independent of the DNA sequence and applied MD protocols, UvrA(2) favors binding of a bent and unwound undamaged DNA, with a kink positioned in the proximity of the Zn3 hairpins, anticollinearly aligned at the bottom of the ventral protein surface. It is further hypothesized that the Zn3 modules play an essential role in the damage recognition process and that the apparent existence of a family of DNA binding sites might be biologically relevant. Our data should prove to be useful in rational (structure-based) mutation studies.     

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1^– cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3′ tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3′ tails after treatment with the rare-cutting endonuclease I-SceI. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3′ tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3′ tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SceI-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SceI-induced double-strand breaks.     

DDB2 (Damaged-DNA binding protein 2) in nucleotide excision repair and DNA damage response

DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.     

Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.     

Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells.

The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.     

Effect of cordycepin(3'-deoxyadenosine) on excision repair of 5,6-dihydroxy-dihydrothymine-type products from the DNA of Micrococcus radiodurans

Cordycepin(3'-deoxyadenosine), a nucleoside analog, has been shown to enhance radiation-induced cell killing. In an effort to elucidate the possible mechanism for enhancement of cell killing, the effect of cordycepin on the excision repair of radiation-induced 5,6-dihydroxy-dihydrothymine-type (t') products from the DNA of wild type Micrococcus radiodurans was investigated. The capacity of M. radiodurans to excise nondimeric (t') products from its DNA was significantly impaired after cordycepin treatment. The results suggest that the increased radiation sensitivity of cordycepin-treated cells could be due to alterations in cellular processes that repair DNA damage.     

The DNA excision repair system of the highly radioresistant bacterium Deinococcus radiodurans is facilitated by the pentose phosphate pathway

Deinococcus radiodurans is highly resistant to radiation and mutagenic chemicals. Mutants defective in the putative glucose-6-phosphate dehydrogenase gene (zwf-) and the aldolase gene (fda-) were generated by homologous recombination. These mutants were used to test the cells' resistance to agents that cause dimer formation and DNA strand breaks. The zwf - mutants were more sensitive to agents that induce DNA excision repair, such as UV irradiation and H2O2, but were as resistant to DNA strand break-causing agents such as methylmethanesulphonic acid (MMS) and mitomycin C (MMC) as the wild-type cells. Analysis of the cytoplasmic fraction of zwf- cells showed that the concentrations of inosine monophosphate (IMP) and uridine monophosphate (UMP) were only 30% of those found in the wild-type cells. The fda- mutants were slightly more resistant to UV light and H2O2. Results suggested that the deinococcal pentose phosphate pathway augmented the DNA excision repair system by providing cells with adequate metabolites for the DNA mismatch repair.     

Single nucleotide patch base excision repair is the major pathway for removal of thymine glycol from DNA in human cell extracts

The repair pathways involved in the removal of thymine glycol (TG) from DNA by human cell extracts have been examined. Closed circular DNA constructs containing a single TG at a defined site were used as substrates to determine the patch size generated after in vitro repair by cell extracts. Restriction analysis of the repair incorporation in the vicinity of the lesion indicated that the majority of TG was repaired through the base excision repair (BER) pathways. Repair incorporation 5' to the lesion, characteristic for the nucleotide excision repair pathway, was not found. More than 80% of the TG repair was accomplished by the single-nucleotide repair mechanism, and the remaining TGs were removed by the long patch BER pathway. We also analyzed the role of the xeroderma pigmentosum, complementation group G (XPG) protein in the excision step of BER. Cell extracts deficient in XPG protein had an average 25% reduction in TG incision. These data show that BER is the primary pathway for repair of TG in DNA and that XPG protein may be involved in repair of TG as an accessory factor.