2. . All genomes constantly being damaged
by UV and other forms of radiation,
chemicals, and other stresses (e.g.,
oxidative, heat).
. Some proteins involved in repair also
function in recombination
e.g., recombination can be used to
repair double-strand breaks.
3. Types of DNA Damage
1. Deamination: (C U and A hypoxanthine)
2. Depurination: purine base (A or G) lost
3. T-T and T-C dimers: bases become cross-
linked, T-T more prominent, caused by UV light
(UV-C (<280 nm) and UV-B (280-320 nm)
4. Alkylation: an alkyl group (e.g., CH3) gets added
to bases; chemical induced; some harmless, some
cause mutations by mispairing during replication
or stop polymerase altogether
4.
5. 5. Oxidative damage: guanine oxidizes to 8-
oxo-guanine, also cause SS and DS
breaks, very important for organelles
6. Replication errors: wrong nucleotide (or
modified) inserted
7. Double-strand breaks (DSB): induced by
ionizing radiation, t,
6. DNA REPAIR
• . To maintain Integrity -mechanisms to
repair damaged DNA. DNA repair -into two
general classes: (1) direct reversal of the
chemical reaction responsible for DNA
damage, and (2) removal of the damaged
bases -replacement with newly synthesized
DNA. Where DNA repair fails, additional
mechanisms -cells to cope with the damage
8. Most damage to DNA is repaired by removal of the damaged bases followed by resynthesis
of the excised region. Some lesions in DNA, however, can be repaired by direct reversal of the
damage, which may be a more efficient way of dealing with specific types of DNA damage that
occur frequently. Only a few types of DNA damage are repaired in this way, particularly
pyrimidine dimers resulting from exposure to ultraviolet (UV) light and alkylated guanine
residues that have been modified by the addition of methyl or ethyl groups at the O6
position
of the purine ring.
UV light is one of the major sources of damage to DNA and is also the most thoroughly
studied form of DNA damage in terms of repair mechanisms. Its importance is illustrated by
the fact that exposure to solar UV irradiation is the cause of almost all skin cancer in humans.
The major type of damage induced by UV light is the formation of pyrimidine dimers, in which
adjacent pyrimidines on the same strand of DNA are joined by the formation of a cyclobutane
ring.
9. Excision Repair
. Most important DNA repair mechanisms in both prokaryotic and eukaryotic cells.
Three types of excision repair—base-excision repair, nucleotide-excision repair,
and mismatch repair.
Uracil can arise in DNA by two mechanisms:
(1) Uracil (as dUTP [deoxyuridine triphosphate]) place of thymine during DNA synthesis,
and
(2) uracil can be formed in DNA by the deamination of cytosine .
10. Base Excision Repair (BER)
Variety of DNA
glycosylases, for different
types of damaged bases.
AP endonuclease
recognizes sites with a
missing base; cleaves
sugar-phosphate
backbone.
Deoxyribose
phosphodiesterase
removes the sugar-
phosphate lacking the
base.
11. Base-excision repair. In this example, uracil (U) has been formed by deamination of cytosine (C)
and is therefore opposite a guanine (G) in the complementary strand of DNA. The bond between
uracil and the deoxyribose is cleaved by a DNA glycosylase, leaving (more...)
The result of DNA glycosylase action is the formation of an apyridiminic or apurinic site
(generally called an AP site) in DNA. Similar AP sites are formed as the result of the spontaneous loss of
purine bases
(see Figure 5.19B), which occurs at a significant rate under normal cellular conditions. For example, each cell
in
the human body is estimated to lose several thousand purine bases daily. These sites are repaired by
AP endonuclease,
which cleaves adjacent to the AP site (see Figure 5.23). The remaining deoxyribose moiety is then removed,
and the
resulting single-base gap is filled by DNA polymeraseand ligase.
12. Mismatch Repair
• Problem: how do cells know which is the right template
strand?
• In E. coli, new DNA not methylated right away
• Mismatch recognized by mutS, then mutL binds and
attracts mutH (endonuclease that cleaves mismatch
and nearest CTAG that is not methylated)
• EuKaryotes (including Arabidopsis) have mutS and mutL
homologues, but no mutH
• Also have the requisite exonucleases, but not clear
how the strand specificity is determined
13. In E.coli, A of each
GATC is methylated.
Mismatch Repair
mutH is endonuclease
14. In E. coli, the ability of the mismatch repair system to distinguish between parental DNA
and newly synthesized DNA is based on the fact that DNA of this bacterium is modified by
the methylation of adenine residues within the sequence GATC to form 6-methyladenine
(Figure 5.25). Since methylation occurs after replication, newly synthesized DNA strands are
not methylated and thus can be specifically recognized by the mismatch repair enzymes.
Mismatch repair is initiated
by the protein MutS, which recognizes the mismatch and forms a complex with two other
proteins called MutL and
MutH. The MutH endonuclease then cleaves the unmethylated DNA strand at a GATC
sequence. MutL and MutS
then act together with an exonuclease and a helicase to excise the DNA between the strand break
and the mismatch
, with the resulting gap being filled by DNA polymerase and ligase.
15. Repair of Double-strand breaks
(DSBs)
2 general ways to repair DSBs:
1. Homologous recombination (HR) - repair of broken DNA using the
intact homologue. Very accurate.
1. Non-homologous end joining (NHEJ) - ligating non-homologous
ends. Prone to errors, ends can be damaged before ligation
(genetic material lost), or get translocations.
18. RecA binds
preferentially to SS DNA
and will catalyze
invasion of a DS DNA
molecule by a SS
homologue.
Important for many
types of homologous
recombination, such as
during meoisis (in
yeast).
DNA glycosylase cuts the bond between base and sugar. AP refers to Apurinic or Apyrimidinic nuclease.
Dam methylase is delayed for about 10 min, then it methylates the new replicon.
Similar to Group I intron homing (in lecture on organellar introns).
Also need a helicase like recQ (occurs in Arabidopsis), a single-strand DNA binding protein (like SSB in E. coli or RepA in humans), and a DNA Ligase. There is also a complex signaling mechanism that couples DSB-type DNA damage to cell cycle progression, basically stopping it if there are any unrepaired DSBs in cells trying to divide. BRCA (breast cancer assoc. gene) is involved in this process, where it functions in loading Rad51 onto the SS DNA extensions.
