In 1964 SETLOW & CARRIER proposed that a damaged base in DNA could be repaired by four enzymatic steps. The damaged base is removed and replaced with a normal base using the complementary strand as a template.
This occurs in four steps:
i) the damaged base is released by a DNA glycosylase-specific for the type of damaged base. This enzyme breaks the bond between the base and sugar.
ii) the abasic sugar (apurinic or apyrimidinic; AP site) is excised by AP endonuclease
iii) DNA polymerase then fills in the missing nucleotide using the undamaged strand as template
iv) the nick is sealed by DNA ligase
Base excision repair is a major pathway for correction of modified bases.
One model proposes five sequential reactions:
i) recognition and removal of the modified base by a specific DNA glycosylase to leave an AP site
ii) incision of the AP site at its 5' side by an AP endonuclease
iii) excision of the 5'-terminal deoxyribose phosphate to leave a single nucleotide gap
iv) DNA synthesis by DNA polymerase I to fill the gap
v) sealing by DNA ligase
An enzyme system hydrolyses two phosphodiester bonds - one on either side of the lesion to generate an oligonucleotide carrying the damage.
The excised oligonucleotide is released from the duplex and the resulting gap is filled in and ligated to complete the repair process.
- the incision patterns are different in eukaryotes and prokaryotes - both hydrolyse the 5th phosphodiester bond on the 3' side of the damage; on the 5' side prokaryotes hydrolyse the 8th phosphodiester bond and eukaryotes the 24th bonds. Hence 12-13 nt are removed in prokaryotes and 27-29 in eukaryotes.
This nuclease activity is termed excinuclease - for excision nuclease - and is unique to DNA repair.
This system repairs CPDs (cyclobutane pyrimidine dimers) and alkylated based without error, but may excise mismatched bases without being able to recognise damaged 'right' from the 'wrong' strand (the one with the misincorporated base) and lead to fixation of the mutation rather than repair. Action of this system on mismatches is inefficient compared to the true mismatch repair system.
Excinuclease activity results frrecognises pyrimidine dimers, a glycosylase
for recognising alkylated bases etc. E.g. MutL,S is a thymine glycosylase
that removes T from C-T mispairs; MutY is an adenine glycosylase that removes
A from A-G and A-C mispairs.
The mechanism is very similar in prokaryotes and eukaryotes, but the proteins share no homology. In E. coli, three proteins UvrA, UvrB and UvrC are necessary and sufficient for excinuclease activity. These are products of the genes: uvrA, uvrB and uvrC, respectively. UvrA is a an adenine triphosphatase, a damage recognition protein and a molecular matchmaker.
i) UvrA and UvrB make an **A2B** complex which binds to the site of the lesion
ii) this complex distorts (unwinds and kinks) the DNA, hydrolysing ATP to provide energy for this process
iii) UvrC binds and UvrB makes the 3' incision that causes a conformational change in the complex, enabling UvrC to make the 5' incision.
iv) Helicase II (UvrD) releases the excised oligomer and UvrC dissociates
v) DNA polymerase I displaces UvrB and fills in the excision gap using the unaltered strand as template.
vi) DNA ligase seals the gap
Fig: nucleotide excision repair
In contrast, the details of human excinuclease are less well understood. At least 17 polypeptides are required, including:
human yeast homolog function
XPF
RAD 1
XPG
RAD 2
XPD
RAD 3
XPC
RAD 4
XPA
RAD 10
damage recognition
ERCC1
RAD 14
XPB
rad 25
- the human repair system has been reconstituted in vitro with highly purified proteins
See: Science vol 266 (23 Dec 1994) pp 1954-56
In E. coli and yeast, mutants unable to carry out NER are sensitive to the mutagenic and lethal effects of UV irradiation. (Hence designations of the repair proteins as RAD.) In humans, xeroderma pigmentosum is associated with defects in NER - hence the XPA to XPG designations of the proteins involved.
There are several pathways of excision repair known. All excision repair systems are believed to use steps 2 to 4. The first step can be brought about in a number of ways. One way is by the action of glycosylase enzymes which recognise different types of damage. For example there is a pyrimidine dimer glycosylase which recongnises pyrimidine dimers, a glycosylase for recognising alkylated bases etc. E.g. MutL, S in thymine glycocylase that removes T from C-T mispairs; MutY is an adenine glycosylase that removes A from A-G and A-C mispairs.
Excision repair is efficient and error-free. Thousands
of UV-induced dimers (induced in the dark) have been shown to be excised
from an E. coli chromosome without error. Rare errors do occur
and may lead to additions or deletions.
DNA polymerases I and III exhibit 3' to 5' exonuclease activities. Their enzymatic action provides the initial step of error correction when a mismatched base pair has been incorporated during DNA synthesis. The 3' to 5' exonuclease activity excises the mismatched base and the polymerase activity inserts the correct base.
In bacterial systems, mismatches may occur at a rate of 1/105 (10-5) nucleotide pairs and proofreading reduces final mismatches by two orders of magnitude to 10-7.
Although the DNA biosynthesis machinery is precise it is not perfect. Mismatch repair mechanism repairs base-pairing errors generated by replication or recombination.
E.g. T opposite G, or a few extra nucleosides added resulting in unpaired bases.
Mismatch repair relies on a system for identifying the template strand and the newly synthesised strand which contain the error. This is provided by adenine methylation at GATC sequence in E. coli. The dam gene codes for a methylase whose target is the adenine in the sequence
5'GATC
3'CTAG
Adenine methylation occurs after DNA strand synthesis, so newly synthesised DNA exists for a while without being methylated. This transient absence of methylation allows the new DNA strand to be targeted for repair.
Methyl-directed mismatch repair involves several proteins, but 4 are critical: MutH, MutL, MutS and MutU.
1) MutS recognises and binds to mismatch
2) MutL binds to mismatched region. This complex brings MutH which has GATC-specific endonuclease activity that cleaves the unmethylated strand at a hemimethylated d(GATC) sequence (only one adenine methylated) 5' or 3' to the misrepair site.
3) The subsequent excision process requires MutS, MutL, DNA helicase II (= MutU = UvrD) and an appropriate exonuclease. A portion of the unmethylated strand spanning the GATC site and the mismatch is removed
4) DNA pol III fills the gap and ligase seals the backbone.
In humans and S. cerevisiae, homologs of bacterial MutS and MutL have been identified, e.g. in humans hMSH2 (MutS); hMLH1, hPMS1, hPMS2 (MutL). Defects at any of these loci are associated with the majority of HNPCC tumors which can lead to cancer.
O6-methylguanine methyltransferase
In E. coli, certain types of DNA damage induced by alkylating agents can be repaired by a single enzymatic step. The most vulnerable site of action of alkylating agents is the base guanine and this base is particularly subject to methylation at the oxygen of carbon atom 6 to give O6-methylguanine. This can mispair with thymine to give GC to AT transitions. This lesion is removed by the enzyme O6methylguanine methylransferase. The enzyme recognises the lesion and the methyl group is transferred to an aa within the enzyme. Apparently there is no way to regenerate the unmethylated enzyme, so a new enzyme is spent for each methyl group removed. The enzyme is encoded by the ada gene in E. coli.
Assume a premutagenic lesion, e.g. a thymine dimer is generated in DNA. If DNA replication starts before the dimer is repaired, replication stalls at the damage and skips past it leaving a gap in the newly synthesised strand. The protein RecA (product of recA in E. coli) binds to single-stranded DNA regions. RecA has the ability to exchange strands between DNA molecules.
The gap opposite the damaged site is filled by recombination of the homologous strand by the normal duplex.
The donor duplex now has a gap that can be filled by repair synthesis or may be fixed later.
E. coli deficient in excision repair and also deficient in RecA (uvr– , recA– ) can only tolerate 1 or 2 thymine dimers, whereas recA+ cells can handle as many as 50 thymine dimers. This implies that RecA protein is very important in DNA repair if excision repair has failed.
This template created by the Web Diner.
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