DNA REPAIR SYSTEMS
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Polynucleotides are fragile and labile, DNA is the most stable of them but is affected by many insulting agents.
Various types of damage:
a. heat - deamination of bases
base loss by glycosylic hydrolysis
b. UV - pyrimidine dimers
6-4 photoproducts
c. ionizing radiation - broken rings
base fragmentation
single stranded breaks
d. chemical modifications - too numerous to
mention
Repair = direct repair (no replacements i.e., base replacement,
nucleotide replacement).
DIRECT REPAIR ( no excision - synthesis - ligation) - usually induced with damage attributed to covalent bond formation - the covalent modification is reversed.
Photolyases - (PL) - complex enzyme molecule - usually has two chromophores associated with the peptide. One is FADH2 the other is either a pterin or a deazaflavin. The enzyme is activated by specific wavelengths of light. Found in procaryotes and eucaryotes. E. coli most studied.
E. coli PL
- from gene phr = 54 kd = monomer
- when isolated looks blue, OD max = 380 nm = FADH2 + pterin
- repairs pyrimidine dimers - 25/min/molecules.
- mutants (phr-) don't photoreactivate and are partly deficient
in excision repair of dimers
- stimulates ABC excinuclease
Recognition:
- binds to pyrimidine dimers as monomers
- very selective - on known sequences having one thymine dimer;
binding only occurs at the dimer.
- minimizes non-specific electrostatic interactions with DNA backbone.
- binds to damage site regardless of DNA form - i.e., dbst, relaxed,
dbst supercoils, ssDNA.
- distortion of the helix apparently not important.
- only recognizes dimers joined via a cis,syn cyclobutane ring
(2 covalent bonds), other dimer structures not bound or repaired.
- PL associates (has footprint of) with 6-7 bp region of the dimer
and only with the strand containing the dimer, in such a way as
to mesh in the minor groove.
Mechanism:
- light at 365-400 activates the enzyme - through both chromophores
- the FADH2 will not work if oxidized to FAD- but binding can
still occur.
- proposed that FAD2 donates electrons, after photoexcitation,
thus cleaving the covalent dimer bonds. The role of the pterin
chromophore may be to donate energy, after excitation, to the
flavin and/or to also donate an electron to the flavin so as to
fill the hole created when the electrons are donated to the dimer.
Note: excitation properties are demonstrated by fluorescence by
the PL.
Yeast PL is similar to E. coli - larger, 66kd.
Same chormophores.
- 50% homology at active sites
- 2 forms PHR1 & PHR2 where PHR2 may be a regulatory gene
for PHR1.
- 365 - 380 nm photoactivates.
BASE EXCISION REPAIR (BER, removal of bases - not the whole nucleotide).
- base is removed by hydrolysis of the N-glycosylic bond between the deoxyribose sugar moiety and the base, by a DNA glycosylase enzyme.
- yields an AP site (i.e.,. apurinic or apyrimidinic site) that must be correctly filled. This is done by nicking the sugar phosphate backbone adjacent to the AP site by an endonuclease. The abasic sugar is removed and a new nucleotide is inserted by polymerase/ligase activity.
- Some enzymes have glycosylase and AP endonuclease
in one molecule.
- Found in procaryotic and eucaryotic cells.
DNA glycosylases:
- usually small, 20-30 kd., narrow substrate specificity
- e.g., specificities include: Uracil, Hypoxanthine, 3-methyladenine
(3-mAde), Formamidopyrimidine, and Hydroxymethyluracil
Uracil occurs due to mis-incorporation or deamination of cytosine by bisulfate, nitrous acids, or spontaneous deamination.
Hypoxanthine occurs due to deamination of adenine by nitrous acids or spontaneous deamination.
3-mAde is created by alkylating agents. E. coli has two 3-mAde glycosylase - TagI and TagII (Gene = tag and alk resp.) (MW = 21 and 31.4 Kd resp.)
Formamidopyrimidine (FAPY) = 7 -mGua - is the most common product of methylating agents of DNA. Gamma radiation produces 4.6-diamino-5-FAPY. E. coli enzyme is from a gene called fpg (30 Kd), cleaves FAPYs.
Hydrorymethyuricil is created by ionizing radiation or oxidative damage to thymidine.
Gylcosylase - AP endonucleases:
Several enzymes have been isolated that appear to do both activities in a concerted way. (i.e., without causing AP site formation) or sequentially.
These include:
Pyrimidine dimer DNA glycosylase - found so far in M. leuteus and T4 infected E. coli - prefers dbst DNA. T4 Endo V = 138 AA = MW 16 kd, makes sequential cleavages - base first, then backbone.
E. coli endonuclease III - also called "redoxyendonuclease", 25 Kd, works only on dbst DNA, concerted cleavage (i.e.,. can't uncouple).
Redoxyendonucleases - several mammalian endos are grouped into this category - have various other names - similar to E coli endo III.
AP Endonucleases:
- cleaves phosphodiester bond 3' or 5' to AP
site
- 4 classes:
I = cleaves 3'--> 3'-OH + 5'-P - and has associated glycosylase
act.
II = cleaves 5' --> 3'-OH + 5'-P
III = cleaves 3' --> 3'-P + 5'-OH
IV = cleaves 5' --> 3'-P + 5'-OH
- those characterized so far:
E. coli exonuclease III = class II AP Endo - in addition to its
3'-->5' exoactivity, it is a major AP Endo for E coli, = 85%
of total act = 28 Kd.
E. coli endonuclease IV = class II AP endo
- 33 Kd - gene = nfo
- mutants very sensitive to alkyating agents (e.g., mitomycin
C & bleomycin)
E coli Endonuclease V - class unknown - 25
Kd
- degrades AP-DNA and DNA with high uracil content.
Human AP Endos - found in HeLa, fibroblasts
and placenta
- several activities isolated - more than one class.
NUCLEOTIDE EXCISION REPAIR
- Damaged bases are removed as oligonucleotides and the resulting gaps are filled with polymerase/ligase activity.
E. coli - best known - ABC excinuclease - ATP
dependent
- 3 subunits - A,B,C (a,b,g) - coded by uvrA, uvrB, and uvrC genes
- cleaves the 8th phosphodiester bond 5' to damaged base and 4th or 5th base 3' to damaged base yielding a 12 - 13 mer fragment. Gap filled by pol-I & ligase.
- genes are not linked - mutants in A&B cannot cleave (incise) but mutants of C can incise.
- recognition of substrate seems to be based on the deformation of the db helical structure. Proposed that a thymine dimer would unwind the helix 19-20 degrees and create a kink of 27 degrees that protrudes into the major grove by 2.6A. Evidence to support the kink and protrusion comes from a slower PAGE electrophoretic mobility of a 64 bp DNA fragment with T-T dimers at 32 base intervals. This slower mobility is eliminated by photoreactivation repair.
- uvrA gene is 2820 bp long ---> 103.8 Kd protein = ATPase activity and DNA binding activity. There are 3 ATPase domains in the AA sequence separated by Zn++ finger domains - thus uvrA protein ligands 2 Zn++. The molecule may be active in dimer form.
- uvrB gene is 2019 bp long ---> 76 Kd peptide (672 AA - no tryptophane) - has ATPase domain homology but no ATPase activity - is monomeric - doesn't bind to DNA alone, only if complexed to uvrA protein -
-uvrC gene is 1764 bp long ---> 66 Kd peptide (588 AA) - is monomeric - can bind to DNA, C-terminus 60 base residues are homologous to human repair gene ERCC-1, some homology with uvrB sequences.
ABC Excinuclease action:
- enzyme assembles sequentially, A2B1 (ATP dependent) complex
first complexes then can bind to damaged DNA or A2 can bind (has
33 bp DNAse I footprint) followed by binding of B yielding a 19
bp footprint (ATP dependent). Indicates either a configurational
change in A2 or release of one A yielding uvrAB-DNA ternary complex.
One or more C subunits combine and excission then occurs.
- ATP:
1. increases dimerization of uvrA - hydrolysis of ATP not needed
though - BAB.
2. is essential for formation of uvrA2B
3. needed for decrease in footprint (above - 33 --> 19).
4. may cause unwinding of helix as much as one turn (360o).
- ABC excinuclease does not become covalently linked to DNA while excising
- Nuclease active site has not been elucidated yet - may be associated with C subunit, but activity not assayed there yet. It may be associated with A dimer with the Zn fingers which may fit into the major grove. B&C addition may force the fingers deeper causing bond separation. Scissions are concerted not sequential.
In human = ERCC = excission repair cross complimenting genes,
i.e., seemed to compliment genes in rodents.
NER gene comparisions:
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RECOMBINATION REPAIR:
In E. coli, if damaged (dimerized) DNA is not repaired before
the replication fork proceeds into the damaged site, the polymerase
holoenzyme stops, dissociates, and reinitiates synthesis about
1000 bp downstream leaving a "gap" with the damaged
bases. RecA protein (present in low constitutive levels) is able
to recombinately transfer the complementary (missing) sequence
from the sister duplex (chromatid) thus creating a gap on the
sister duplex that has an undamaged template. The crossing over
is staggered such that a small gap remains downstream of the dimer
site. DNA pol-I is then able to switch templates to fill both
gaps. Ligase finishes the job. Note, however, the dimer is still
present but can be repaired by the other mechanisms.
SOS REPAIR:
- from sos genes of a "regulon" = 20 genes.
- LexA protein is a negative repressor of these genes.
It binds to the "sos box" (5'-CTG-N10-CAG-3') consensus
sequence that overlaps the genes promoter regions.
- severe damage (eg. UV light) causes ssDNA (gaps)
recA protein, which is constitutive, binds to ssDNA and associates
with the lexA protein causing it to become activated (config change?)
in such a way that it performs autocleavage (i.e., of itself).
- the denatured lexA protein dissociates thereby removing the
repression of the genes including:
lexA, recA (repressor & repair resp.)
uvrA + uvrB + uvrC (= excission repair)
umuDC (damage bypass repair - guesses, error prone)
sulA, sulB (cell division regulators)
din genes, recN, recQ (functions unknown) + others.
- cell division is inhibited to allow more
time for repair
- causes "error prone repair" in that the missing sequences
are replaced by "guessing". Unreliable templates = "sinking
ship". This increase in mutation may promote survival by
trying anything.
MITOCHONRIAL DNA REPAIR:
- mtDNA seems more vulnerable to damage. Less redundancy of genes
would lead to expect excellent repair. Not so.
- damage results from high oxidative environment (from ox phos)
plus all other sources (chemical, radiation, etc.). oxidative
damage measured by formation of 8-OH-dG. Also find abasic sites,
misincorporated bases, base modifications (which cause mismatches
at replication), T & C lesions, and FAPY.
- early studies with human cell mito indicated
that thymine dimers were not removed thus suggesting a lack of
NER.
- other studies have recently found a variety of repair activities.
These include methyl transferase, alkyl transferase, uracil DNA
gycosylase, AP endonuclease (stimulated by asbestos in human cells),
UV endonuclease, and UV photoreactivation activity (photolyase?).
- Much of this suggests BER and Photoactivation repair.
- In human, 2 UDGs (1 & 2) were found, UDG2 is 36kd and is
nuclear while UDG1 is 30 kd and is mitochondial in location. UDG1
may be a cleavage product (PTLM) of UDG2 and transported into
the mito. Also, UV light forms cyclobutane pyrimidine dimers (CPD)
and (more so) 4-6-pyrimidine photoproducts (4-6 PP). Neither are
repaired in human mitochondria. Some mammalian mitochondrial extracts
have shown recombination activities but not demonstrated in intact
mitochondra.
- Xenopus mito lysates have shown increased DNA synthesis activity
in response to H2O2, acid and UV. Suggests BER. UV damage is repaired
in the presence of light. Photoactivation is also found in Maize.
- Yeast have a protein, MSH1, that specifically binds to mtDNA
at mismatched sites. Also has a site for ATPase activity.
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