base excision repair
Thursday 20 November 2003
Definition: Base excision repair (BER) protects against damage to DNA from reactive oxygen species, methylation, deamination, hydroxylation and other by-products of cellular metabolism.
The survival of organism depends on the accurate transmission of genetic information from one cell to its daughters. Such faithful transmission requires not only extreme accuracy in replication of DNA and precision in chromosome distribution, but also the ability to survive spontaneous and induced DNA damage while minimizing the number of heritable mutations.
To achieve this fidelity, cells have evolved surveillance mechanisms that monitor the structure of chromosomes and coordinate repair and cell cycle progression. BER (Base Excision Repair) is the main guardian against the most frequent types of DNA damage due to cellular metabolism, including that resulting from ROS (Reactive Oxygen Species), methylation, deamination, hydroxylation or spontaneous loss of the DNA base itself.
These alterations, although simple in nature, are highly mutagenic and therefore represent a significant threat to genome fidelity and stability.
The BER system
BER involves the concerted effort of several repair proteins that recognize and excise specific DNA damages, eventually replacing the damaged moiety with a normal nucleotide and restoring the DNA back to its original state. BER has two subpathways, both of which are initiated by the action of a DNA Glycosylase.
In humans, six DNA Glycosylases have been identified, and each excises an overlapping subset of either spontaneously formed (e.g. hypoxanthine), oxidized (e.g. 8-oxo-7, 8-dihydroguanine), alkylated (e.g. 3-methyladenine), or mismatched (e.g. T: G) bases.
Such DNA Glycosylases bind specifically to a target base and hydrolyze the N-glycosylic bond, releasing the inappropriate or damaged base while keeping the sugar phosphate backbone of the DNA intact.
This cleavage generates an AP (Apyrimidinic/Apurinic) or Abasic site (i.e. the site of base loss) in the DNA. Alternatively, AP sites also arise by the spontaneous hydrolysis of the N-glycosidic bond.
In either case, the AP site is subsequently processed by APE1 (AP Endonuclease-1, also called HAP1/REF1/ APEX), which cleaves the phosphodiester backbone immediately 5 to the AP site, resulting in a 3 hydroxyl group and a transient 5’ dRP (abasic Deoxyribose Phosphate).
APE1 is the predominant AP Endonuclease in mammalian cells. Removal of the dRP is accomplished by the action of DNA PolBeta (Polymerase Beta), which adds one nucleotide to the 3’ end of the nick and removes the dRP moiety via its associated AP Lyase activity. The strand nick is finally sealed by a DNA Ligase, thus restoring the integrity of the DNA.
Replacement of the damaged base with a single new nucleotide is referred to as "short-patch" repair and represents approximately 80-90% of all BER. PolBeta also interacts with the noncatalytic XRCC1 subunit of the XRCC1-DNA Ligase-III heterodimer.
Consequently, XRCC1 acts as a scaffold protein by bringing the Polymerase and Ligase together at the site of repair; further stabilization of the complex may be achieved by direct binding of the NH2-terminal region of XRCC1 to the DNA single-strand break.
In cases where the terminal sugar-phosphate residue has a more complex structure that is relatively resistant to cleavage by the AP Lyase function of PolBeta, DNA strand displacement occur, involving either PolBeta or a larger Polymerase such as PolDelta, for filling-in of gaps a few nucleotides long.
This represents the back-up pathway of BER, termed "long-patch" repair. PARP (Poly ADP Ribose Polymerase), which binds to and is activated by DNA strand breaks, and the recently identified PNK (Polynucleotide Kinase) is important when BER is initiated from a single strand break to protect and trim the ends for repair synthesis.
The FEN1 (Flap Endonuclease-1) structure-specific nuclease removes the displaced dRP as part of a "flap" oligonucleotide prior to sealing of the nick by a DNA Ligase and the PCNA (Proliferating Cell Nuclear Antigen) protein stimulates these reactions, acting as a scaffold protein in this alternative pathway in a way similar to that of XRCC1 in the main pathway.
Another replication factor, DNA Ligase-I then completes this longer patch form of repair. In addition to the processing of 5’ ends of Okazaki fragments during lagging-strand DNA replication, FEN1 minimizes the possibility of hairpin loop formation and slippage during strand displacement and subsequent DNA synthesis, which might otherwise result in local expansion of sequence repeats.
Long-patch repair results in the replacement of approximately 2-10 nucleotides including the damaged base. Temporary inefficiency in this process during early mammalian development results in several human syndromes such as Huntingtons disease, which are associated with expansion of triplet repeats in relevant genes.
Long-term effects result from irreversible mutations contributing to oncogenesis. The inability to repair DNA damage properly in mammals leads to various disorders and enhanced rates of tumor development.
No human disorders caused by inherited BER deficiencies have been identified, but inactivation of BER core proteins induces embryonic lethality, highlighting the vital importance of the process as a whole.
This might be due to the contribution of spontaneously occurring Abasic sites and single strand breaks that directly feed into the BER core reaction and/or to the generation of reaction intermediates by the Glycosylases that cannot be further processed.
The outcome of DNA damage is diverse and generally adverse. Acute effects arise from disturbed DNA metabolism, triggering cell-cycle arrest or cell death.
ROS-associated DNA lesions
Reactive oxygen species (ROS) from endogenous and environmental sources induce oxidative damage to DNA, and are a threat to the integrity of genome. This oxidative DNA damage can be restored by the base excision repair (BER) pathway that is conserved from bacteria to humans.
Active oxygen species in the nucleotide pool of the cell can produce 8-oxo-dGTP (8-oxo-7,8-dihydrodeoxyguanosine triphosphate), which can then be incorporated into cellular DNA. Human cells contain enzyme activity that hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, thereby preventing occurrence of mutations caused by misincorporation.
This oxidative DNA damage is restored by the base excision repair (BER) pathway that is conserved from bacteria to humans and is initiated by DNA glycosylases, which simply remove the aberrant base from the DNA backbone by hydrolyzing the N-glycosidic bond (monofunctional DNA glycosylase), or further catalyze the incision of a resulting abasic site (bifunctional DNA glycosylase).
In eukaryotic cells, DNA polymerase beta (POLB) performs base excision repair (BER) required for DNA maintenance, replication, recombination, and drug resistance. Two important enzymatic steps in mammalian BER are contributed by POLB: DNA resynthesis of the repair patch and lyase removal of the 5-prime-deoxyribose phosphate.
BER is initiated by a class of DNA-repair-specific enzymes - DNA glycosylases - each of which recognizes a single or a small subset of chemically altered or inappropriate bases.
For example, an enzyme called uracil DNA-glycosylase specifically recognizes uracil as an inappropriate base in DNA and catalyses hydrolysis of the N-glycosyl bond that links the uracil base to the deoxyribose-phosphate backbone of DNA.
Uracil is thus excised from the genome as a free base, leaving a site of base loss in the DNA - an apyrimidinic site in the case of uracil removal, or an apurinic site when a purine is lost. These so-called AP (or abasic) sites are repaired by a further series of biochemical events.
The BER pathway
apurinic/apyrimidinic endonuclease/redox factor 1 (APE1/ref1)
base-excision repair diseases
- BER DNA glycosylase MYH biallelic mutations in autosomal recessive syndrome of adenomatous colorectal polyposis
Farrington SM, Tenesa A, Barnetson R, Wiltshire A, Prendergast J, Porteous M, Campbell H, Dunlop MG. Germline Susceptibility to Colorectal Cancer Due to Base-Excision Repair Gene Defects. Am J Hum Genet. 2005 May 3;77(1). PMID: 15871140
Fortini P, Pascucci B, Parlanti E, D’Errico M, Simonelli V, Dogliotti E. The base excision repair: mechanisms and its relevance for cancer susceptibility. Biochimie. 2003 Nov;85(11):1053-71. PMID: 14726013
Cheadle JP, Sampson JR. Exposing the MYtH about base excision repair and human inherited disease. Hum Mol Genet. 2003 Oct 15;12 Spec No 2:R159-65. PMID: 12915454