Deficiency in repair of nuclear and mitochondrial DNA damage has been linked to several neurodegenerative disorders. Many recent experimental results indicate that the post-mitotic neurons are particularly prone to accumulation of unrepaired DNA lesions potentially leading to progressive neurodegeneration. Nucleotide excision repair is the cellular pathway responsible for removing helix-distorting DNA damage and deficiency in such repair is found in a number of diseases with neurodegenerative phenotypes, including Xeroderma Pigmentosum and Cockayne syndrome. The main pathway for repairing oxidative base lesions is base excision repair, and such repair is crucial for neurons given their high rates of oxygen metabolism. Mismatch repair corrects base mispairs generated during replication and evidence indicates that oxidative DNA damage can cause this pathway to expand trinucleotide repeats, thereby causing Huntington's disease. Single-strand breaks are common DNA lesions and are associated with the neurodegenerative diseases, ataxia-oculomotor apraxia-1 and spinocerebellar ataxia with axonal neuropathy-1. DNA double-strand breaks are toxic lesions and two main pathways exist for their repair: homologous recombination and non-homologous end-joining. Ataxia telangiectasia and related disorders with defects in these pathways illustrate that such defects can lead to early childhood neurodegeneration. Aging is a risk factor for neurodegeneration and accumulation of oxidative mitochondrial DNA damage may be linked with the age-associated neurodegenerative disorders Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. Mutation in the WRN protein leads to the premature aging disease Werner syndrome, a disorder that features neurodegeneration. In this article we review the evidence linking deficiencies in the DNA repair pathways with neurodegeneration.
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Fig. 1. DNA lesions and their repair by the four major DNA repair pathways in higher eukaryotes
Cells have multiple DNA repair pathways that provide the capacity to repair many different types of DNA lesions. The figure provides an overview of DNA damaging agents, the lesions they cause and the four main pathways responsible for removing and repairing the DNA lesions.
Fig. 2. The two subpathways of mammalian nucleotide excision repair
In global genome nucleotide excision repair (GG-NER) helix distorting DNA damage anywhere in the genome is recognized by the XPC-HR23B-CEN2 complex. The DDB complex consisting of the two subunits DDB1 and DDB2 (XPE) can facilitate recognition of lesions that by themselves cause little distortion of the helix. In transcription-coupled nucleotide excision repair (TC-NER) recognition is by the stalling of RNA pol II at DNA lesions on the transcribed strand of active genes facilitated by CSB, CSA and XAB2. Either XPC in GG-NER or CSB and CSA in TC-NER recruit TFIIH to the repair site followed by converging of the subpathways. The XPB and XBD subunits of TFIIH are DNA helicases that unwind the DNA in the immediate vicinity of the lesion. RPA and XPA bind to keep the DNA strands apart. For the dual incision, XPA recruits the XPF-ERCC1 endonuclease to incise the damaged DNA strand 5′ to the lesion while XPG incises 3′ to it. The lesion is thus excised in an oligonucleotide fragment leaving behind a single-strand gap. Repair synthesis is performed by DNA polymerase δ and κ, or ε (Pol δ/κ/ε) with the help of the accessory proteins RFC, PCNA and RPA. The remaining nick in the DNA backbone is sealed with ligation by either LIG1 or LIG3α-XRCC1.
Fig. 3. The mammalian base excision repair and single-strand break repair pathways
Base excision repair (BER) is initiated by removal of the modified base by either a monofunctional or bifunctional DNA glycosylase to leave an abasic site (AP). If excision is by either one of the monofunctional DNA glycosylases UDG or MPG, the following incision of the DNA backbone 5′ to the AP site is by APE1. Excision by one of the bifunctional DNA glycosylases NTH1, OGG1, NEIL1 or NEIL2 is followed by incision 3′ to the AP site via β- or βδ-elimination facilitated by the intrinsic 3′ AP lyase activity of these enzymes. The resulting single-strand break will contain either a 3′ or 5′ obstructive termini. End processing is then performed by Pol β, APE1 or PNKP depending on the specific nature of the terminus. Single-strand breaks do not only occur as intermediates of BER but also by other means and can contain simultaneous 3′ and 5′ obstructive termini. PARP1 recognizes these breaks and the end processing may utilize the additional factors TDP1 and APTX. When end processing has produced the necessary 3′-OH and 5′-P termini the following BER and single-strand break repair (SSBR) steps diverge into two subpathways, short-patch and long-patch. In short-patch BER/SSBR repair synthesis of the single nucleotide gap is by Pol β aided by the XRCC1 scaffold, and subsequent ligation by LIG3α finishes the repair. In long-patch BER/SSBR repair synthesis of the 2–13 nucleotide gap is by Pol β, and/or Pol δ/ε aided by PCNA and RFC. A resulting 5′ flap is removed by FEN1 and the the final ligation step is by LIG1.
Overview of studies linking BER and neurodegeneration.
Fig. 5. Human mismatch repair
For convenience, the mechanism of human mismatch repair (MMR) can be seen as consisting of five consecutive steps: (i) Recognition and binding of a mismatch is by either a MSH2-MSH6 or MSH2-MSH3 heterodimeric ATPase complex. MSH2-MSH6 preferentially recognizes base-base mismatches and insertion deletion loops of 1–2 nucleotides while MSH2-MSH3 has preference for larger insertion-deletion loops. The mismatch-bound MSH2-MSH6 (or MSH2-MSH3) recruits the MLH1-PMS2 complex, a molecular matchmaker with weak ATPase activity, to form a ternary complex. The PCNA clamp recruits MMR proteins to the replication fork while the clamp loader RFC loads PCNA. A strand-specific nick or gap, which may reside either 5′ or 3′ to the mismatch, is sufficient to direct repair in 5′- and 3′-directed MMR, respectively. PCNA appears essential for 3′-directed but not 5′-directed MMR. (ii) Excision is apparently by the 5′ to 3′ exonuclease EXO1 in both 3′- and 5′-directed MMR. For 5′-directed MMR the excision is straightforward by the 5′ to 3′ exonuclease activity of EXO1. For 3′-directed MMR, the endonuclease function of PMS2 is activated by presence of the 3′ nick, and stimulated by RFC, PCNA and ATP, to introduce a necessary second nick 5′ to the mismatch. Excision can then follow by EXO1. RPA binds to protect the single-stranded DNA during the excision and to facilitate the following DNA repair synthesis. (iv) Repair synthesis is accurately performed by Pol δ. (v) Ligation of the remaining nicks after synthesis is by LIG1.
Fig. 6. Model for the generation of single-strand breaks from TOP1 cleavage complexes
During various processes of DNA metabolism the enzymatic activity of DNA topoisomerase I (TOP1) generates reversible 3′-TOP1-DNA intermediates known as TOP1 cleavage complexes. Such complexes can, however, become unduly long lived and collision with RNA pol II or the proximity of a DNA lesion creates a TOP1-associated DNA single-strand break. The enzyme responsible for cleaving the link between TOP1 and the 3′-teminus of the single-stranded break is TDP1. If excision is successful the remaining strand break can then be repaired by a SSBR complex consisting of PNKP, XRCC1, Pol β and LIG3α thereby restoring DNA integrity. If excision by TDP1 fails, a persistent DNA single-strand break will be generated.
Fig. 7. Model for the differential impact of single-strand breaks on dividing cells and post-mitotic neurons caused by AOA1 or SCAN1 mutations, or aging
The upper part of the figure shows the various 3′- and 5′-obstructive termini and the proteins responsible for resolving them. The lower part of the figure shows the impact of single-strand breaks on wild-type, AOA1 and SCAN1 cells. In both dividing and post-mitotic wild-type cells efficient single-strand break repair (SSBR) will ensure resolution of the break. In dividing AOA1, SCAN1 or aging cells SSBR is deficient, but the single-strand breaks may be converted to double-strand breaks and subsequently repaired by homologous recombination (HR). In the post-mitotic neurons of AOA1 or SCAN1 patients, or aging individuals HR is not available and persistent unrepaired single-strand breaks in these cells lead to neuronal cell death.
Fig. 8. The two major mammalian pathways for double-strand break repair
Repair of a DNA double-strand break (DSB) is usually accomplished by either homologous recombination (HR) when a homologous chromosome is available in the form of a sister chromatid, or by non-homologous end-joining (NHEJ) throughout the cell cycle. Following formation of a DSB, the MRE11-RAD50-NBS1 (MRN) complex is recruited to the break where it binds to the DNA ends through MRE11. A coiled-coil region of RAD50 reaches across the break and through coordination of a central Zn
2+ ion, tethers the two broken DNA ends together. The serine/threonine protein kinase ATM is usually present as an inactive dimer but is recruited to the break site by NBS1. This causes ATM to autophosphorylate (P) and monomerize, activating the kinase. The activated ATM phosphorylates NBS1 and a multitude of other proteins that participates in the DNA DSB response, including BRCA1 and phosphorylation of nearby histone H2AX to generate γH2AX. The γH2AX at repair foci acts as a signal to recruit other repair proteins in order to assemble DSBR complexes. One such protein, MDC1, binds to γH2AX which recruits RNF8to the break site where it initiates an ubiquitylation cascade of histones H2A and H2AX causing chromatin restructuring and generation of binding sites for further protein factors. The HR pathway proceeds via the end processing of damaged DNA termini with initial nucleolytic 5′ resection performed by CtIP, a function dependent on CtIP recruitment of BRCA1. Further resection by EXO1 generates single DNA strands with 3′ overhangs upon which RAD51 monomers attach to form nucleoprotein filaments. A RAD51 recombinase complex is then assembled containing the accessory proteins BRCA1, BRCA2, RAD52 and the RAD51 paralogs: RAD51B/C/D and XRCC2/3. This recombinase complex, with the aid of an additional factor, RAD54, facilitates homology search and strand invasion of the homologous chromosome to form displacement loops (D-loops). DNA repair synthesis by DNA polymerase using the homologous strand as template extends the 3′ invading strand allowing branch migration of the Holliday junction, a cruciform intermediate. The repair synthesis allows capture of the other DNA end to create a double Holliday junction intermediate. DNA ligation by LIG1 seals the remaining nicks and resolution of the double Holliday junction by structure-specific endonucleases generate either non-crossover or crossover products depending on where the junctions are cut by the resolvase. The BLM RecQ helicase can cooperate with TOPO3α and BLAP75 to resolve DHJs with generation of exclusively non-crossover products. The NHEJ pathway first involves binding of Ku70-80 heterodimer to the two DNA termini. This then recruits DNA-PK CS to form the DNA-PK complex bringing the two DNA termini close together. Because of associated lesions not all DNA termini are readily ligatable and must be processed to generate the 5′-P and 3′-OH termini that are necessary for ligation. Autophosphorylation of DNA-PK CS can make the obstructive termini accessible to end processing enzymes such as TDP1. DNA-PK CS also mediates a regulatory phosphorylation of the WRN RecQ helicase. End processing and resection, while not fully understood, may involve the exonuclease activity of FEN1, WRN and Artemis. DNA polymerases then perform any necessary DNA repair synthesis. Finally, the DNA-PK complex, in a Ku70-80 mediated fashion, recruits the LIG4-XRCC4-XLF complex to perform the ligation of the DNA termini via the ligase activity of LIG4.
Overview of DNA repair pathways active in the nucleus and mitochondria of mammalian cells.
Fig. 10. Model for mammalian inter-strand crosslink repair
The ICL repair process is influenced by cell-cycle status. (A) In ICL repair in non-replicating cells the crosslink is recognized by the XPC-HR23B-CEN2 complex or RNA pol II. The XPF-ERCC1 complex makes incisions in the DNA on either side of the ICL leaving a gap in the opposing strand and the incised oligonucleotide still attached to the intact strand. TLS polymerases fill the gap while the flipped out crosslinked base is recognized by the DBB1-DDB2 complex, triggering the completion of repair by NER. (B) In actively replicating cells ICL repair at stalled replication forks is initiated when the arrested fork activates ATR and its downstream kinase CHK1. Facilitated by RPA and the MRN complex, ATR and CHK1 phosphorylates many proteins of the FA-BRCA network. The FANCM-FAAP24 complex enables access of other repair proteins, and subsequently becomes part of the FA core complex. The FA core complex monoubiquitylates the FANCD2-FANCI complex, which is then retained at the damaged region by BRCA1. The ICL is unhooked by XPF-ERCC1 and MUS81-EME1, leaving a DSB. The monoubiquitinated FANCD2-FANCI complex releases the replicative polymerase Pol δ and loads an error-prone TLS polymerase to perform bypass synthesis to repair the gap, possibly assisted by FANCJ. The crosslinked base is removed by NER. After 5′ end resection, possibly by the MRN complex, the BRCA2-FANCN-RAD51 complex initiates the reconstruction of the replication fork by HR.
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Research Support, Non-U.S. Gov't
DNA Repair-Deficiency Disorders / genetics
DNA Repair-Deficiency Disorders / metabolism
DNA Repair-Deficiency Disorders / physiopathology*
Neurodegenerative Diseases / etiology
Neurodegenerative Diseases / genetics*
Neurodegenerative Diseases / metabolism
Reactive Oxygen Species / metabolism