Lecture 7: DNA Repair Mechanisms

Microbial  Gene-cs  
BI  302  
Dr.  Nalina  Nadarajah  
Email:  nnadarajah@centennialcollege.ca  
 Agenda  for  This  Week  
1. Mechanisms  that  Reverse  DNA  Damage  
– photoreac-va-on  
– methyltransferase  
2. Mechanisms  that  Excise  DNA  Damage  
– Nucleo-de  excision  repair  (NER)  
– Mismatch  repair  
– Very  short  patch  (VSP)  repair  
– Glycosylases  
3. Mechanisms  that  Tolerate  DNA  Damage  
– Transdimer  synthesis  
– Post  replica-on/  recombina-on  (PRR)   2  
 Introduc-on  to  DNA  Repair  
• The  integrity  and  stability  of  DNA  are  essen-al  to  life  
• However,  it  is  constantly  under  assault  from  radia-on,  
mutagens,  spontaneous  changes  
• If  DNA  no  repair  mechanisms  -­‐-­‐>  cells  ability  to  survive  
would  be  compromised  
• If  DNA  repair  mechanisms  are  too  efficient  -­‐-­‐>    also  problem  
–  why?  
• The  rate  of  muta-on  is  low  due  to  efficient  repair  
mechanisms  
3  
 What  is  DNA  Damage  
• DNA  is  damaged  when  
– the  sequences  of  the  bases  changed  
– phosphate  backbone  changed  or  distorted  

• Each  change  in  DNA  is  described  as  a  lesion  

– lesions  impact  damaged  DNA’s  ability  to  convey  its  precise  
sequence  of  nucleo-des  to  future  daughter  cells  
– less  than  1  in  1000  DNA  lesion  becomes  a  muta-on  

4  
Damage  to  DNA  Occurs  in  Response  to  
Mutagens    
• Mutagenic  chemicals  include  
– base  analogues  (similar  in  structure  to  the  normal  bases  and  
can  become  incorporated  into  DNA)  

– base-­‐modifying  agents  (which  can  change  a  base)  

– intercala-ng  agents  (cause  inser-ons  and  dele-ons)  

• UV  radia-on  (sunlight)  can  cause  pyrimidine  dimer  

forma-on  -­‐-­‐>  block  replica-on  and  transcrip-on  

• Ionizing  radia-on  (such  as  X-­‐rays)  generates  highly  reac-ve  

intermediates  that  causes  ss  and  ds  breakage   5  
 DNA  Damage  and  Causa-ve  Agents  
DNA  Damage   Causa,ve  Agent  
Pyrimidine  dimers   UV  light  

Single  and  double  stranded  breaks   Ionizing  radia-on,  endonucleases,  

peroxides  

Mismatched  base  pairs   Incorpora-on  errors,  tautomeric  

shics,  spontaneous  deamina-on,  
base  analogs  

Covalent  crosslinks   UV  light,  ionizing  radia-on,  HNO2  

Apurinic  and  apyrimidinic  sites   Spontaneous  hydrolysis,  alkyla-ng  

agents  
6  
Major  Types  of  Lesions  

7  
 Types  of  Double  stranded  Breaks  

What happens when your DNA is

damaged? Animation

• Blunt  –  break  on  both  strands  at  the  same  point  

• Staggered  –  break  at  different  loca-ons  à  s-cky  ends  
8  
Reverse,  Excise  or  Tolerate?  
• Mostly  either  reverse  or  
excise  
• several  repair  mechanisms  
can  dispose  similar  lesions  
• e.g.  pyrimidine  dimers  
• Func-ons  before  DNA  
replica-on  
• If  damaged  DNA  gets  
replicated,  another  group  
of    mechanisms  can  
tolerate  the  lesions  
– so  replica-on  can  proceed  
through  
– lesions  NOT  actually  
repaired   9  
 Mechanisms  that  Reverse  DNA  
Damage  
Least  error  prone  because  no  new  DNA  synthesis  involved  
1. Photo-­‐reac-va-on  
– first  reported  in  1949  by  Albert  Kelner  
– when  Streptomyces  griseus  was  irradiated  with  a  large  dose  
of  UV,  chance  of  survival  increased  when  exposed  to  visible  
light  (blue  range)  
2. Methyl-­‐transferase  
– tackles  lesions  arising  from  alkyla-on  

10  
 1. Photoreac-va-on  

• UV  light  creates  pyrimidine  dimers  

• Can  be  par-ally  reversed  by  exposing  to  blue  light  
• Depends  on  the  ac-vity  of  photoreac-va-on  enzyme  (PRE)  
aka  photolyase  
• 3  step  process  to  repair  cyclobutane  dipyrimidine  lesions  

11  
Photoreac-va-on  
3  steps:  
1. Enzyme  scans  for  cyclobutane  
dipyrimidine  lesions  
– when  found  one,  it  binds  to  the  
lesion  
2. The  photolyase-­‐lesion  complex  
absorbs  a  quantum  of  light  
(350-­‐500  nm)  à  uncoupling  of  
pyrimidine  dimer  
3. Photolyase  is  released  from  DNA;  
resumes  scanning  

Animation 12  
 Photoreac-va-on  Cont.  

• Photoreac-va-on  is  not  absolutely  essen-al  in  E.coli  

– muta-on  in  gene  coding  for  PRE  is  not  lethal  

• Error-­‐free  because  excision  or  DNA  synthesis  not  required  

• Photoreac-va-on  has  been  iden-fied  in  bacteria,  fungi,  

plants  and  some  vertebrates,  but  NOT  in  humans  
– humans  rely  on  other  repair  mechanisms  to  reverse  the  
effects  of  UV  radia-on  

13  
 2. Methyltransferases  
• Also  known  as  alkyltransferase  
• Tackles  lesions  arising  from  alkyla-on  
• e.g.  O6-­‐methylguanine  or  O4-­‐methylthymine  
methyltransferase  
• When  DNA  is  alkylated  by  nitrosoguanidine  (NTG),  the  most  
vulnerable  bases  are  G  (primarily)  and  T  (occasionally)  
– give  rise  to  O6-­‐methylguanine  and  O4-­‐methylthymine  

14  
O6-­‐methylguanine  methyltransferase  
3  step  process:  
1. The  ada  gene  product,  
methyltransferase  scans  DNA  
for  O6-­‐methylguanine  and  
binds  to  the  lesion  
2. methyl  or  other  alkyl  group  
transferred  from  guanine  to  
ada  methyltransferase  
– upon  removal  of  alkyl  group,  
G  and  T  resume  their  normal  
bonding  behaviour  
3. methyltransferase  
inac-vated  and  degraded  
15  
O6-­‐methylguanine  methyltransferase  
• The  ada  methyltransferase  can  also  remove  alkyl  groups  
from  the  phosphate  backbone  
• Not  true  enzymes  
– act  only  as  acceptors  
– do  not  catalyze  reac-ons  
• Repair  system  can  be  saturated  if  alkyla-on  is  too  high  

Mechanisms that Reverse DNA Damage 16  

Mechanisms  that  Excise  DNA  Damage  

1. Nucleo-de  excision  repair  (NER)  

2. Mismatch  repair  
3. Very  short  patch  (VSP)  repair  
4. Base  Excision  Repair  (BER)  by  Glycosylases  

17  
1. Nucleo-de  Excision  Repair  System  
• First  reported  by  Robert  Setlow  in  1964  
• Referred  to  as  “dark  repair”  
• Non-­‐specific  –  can  repair  several  types  of  lesions  
• NER  system  recognized  major  distor-ons  (i.e.  bulky  lesions)  
not  minor  distor-ons  such  as  mismatched  base  pairs,  O6-­‐
methylguanine,  O4-­‐methylthymine  etc.  
• Found  in  all  organisms  from  bacteria  to  humans  

18  
19  
 Uvr  ABC  directed  NER  
Two  Models:  
• Model  1  
– Uvr  ABC  endonuclease  recognizes    the  distor-on  and  makes  
an  incision  5’  to  the  dimer  on  the  same  strand  as  dimer  
– incision  cuts  the  phosphodiester  backbone  
– the  3’  to  5’  exonuclease  ac-vity  of  DNA  pol  I  removes  7-­‐20  
nucleo-des  from  the  cut  strand  including  the  dimer  
– DNA  pol  I  uses  5’  to  3’  polymerizing  ac-vity  to  replace  the  
missing  nucleo-des  
• uncut,  undamaged  complementary  strand  as  template  
• removal  and  polymeriza-on  occurs  simultaneously  
20  
– Ligase  seals  the  remaining  ss  break    
• Model  2:  
– upon  recogni-on,  2  incisions  on  the  same  strand  are  made  
• 5’  and  3’  to  the  dimer  
– incision  releases  the  damaged  DNA  
– no  need  to  use  DNA  pol  I’s  3’  to  5’  exonuclease  ac-vity  
– DNA  pol  I  replaces  missing  DNA  with  polymeriza-on  ac-vity  
– ligase  seals  the  ss  break  
Animation of Model 2

Differences  between  the  two  models:  

a) number  of  incisions  
b) loca-on  of  the  incision  
c) the  need  for  exonuclease  ac-vity   21  
Nucleo-de  Excision  Repair  Mechanism  
• Damage  to  the  DNA  distorts  config.  
enzyme complex
• An  enzyme  complex  recognizes  the  
distor-on  
ss binding proteins
• The  DNA  strands  are  separated;  
single  strand  binding  proteins  
stabilize  ss  
• An  enzyme  cleaves  the  strand  on  
both  sides  of  the  damage  
• Part  of  the  damage  strand  is  
removed  
• The  gap  is  filled  by  DNA  
polymerase  and  sealed  by  ligase  
22  
 2. Mismatch  Repair  

• Replica-on  is  extremely  accurate  

– each  new  copy  has  less  than  1  error  per  106  nucleo-des  

– most  errors  are  corrected  by  proof  reading  (3'-­‐5'  exonuclease  

ac-vity  of  DNA  Pol)  and  never  become  permanent  muta-ons  

– many  incorrectly  inserted  that  escaped  proof  reading  is  

repaired  by  mismatch  repair  

23  
 MutHLS  methyl  directed  Mismatch  
Repair  (MMR)    
• The  proof  reading  of  DNA  pol  does  not  always  correct  
mismatch  
• Mismatch  Repair  is  required  to  repair  errors  that  escape  
proof-­‐reading  systems  during  DNA  replica-on  
• MutHLS  methyl  directed  MMR  is  a  major  excision  repair  
mechanism  in  E.coli  
• Efficient  mechanism  based  on  the  fact  that  parental  
template  is  methylated  and  newly  synthesized  daughter  
DNA  is  not     24  
 Principle  Behind  MMR  
• These  repair  systems  can  dis-nguish  newly-­‐synthesized  DNA  
from  parental  DNA  
– newly-­‐synthesized  strands  are  non-­‐methylated  while  parental  DNA  
strands  are  methylated  

• Methyla-on  of  DNA  is  due  to  the  ac-vity  of  the  Dam  (DNA  
adenine  methylase)  methylase  which  methylates  adenine  
bases  within  the  sequence  GATC  
• Dam  methylase  adds  a  methyl  group  to  the  adenine  of  the  
sequence  5'-­‐GATC-­‐3’  in  newly  synthesized  DNA  
– immediately  acer  DNA  synthesis,  the  daughter  strand  remains  
unmethylated  for  a  short  -me  
25  
 MutHLS  directed  MMR  
In DNA replication mismatch
base added to new strand

• methyla-on  at  GATC  allows  old  and  

new  strands  to  be  differen-ated  
– lag  in  methyla-on  
– immediately  acer  replica-on,  the  old  
strand  methylated  &  new  strand  not  
• mismatch  repair  complex  (blue)  brings  
the  mismatch  bases  close  to  the  
methylated  GATC  sequence  &  the  new  
strand  is  iden-fied  
• Exonuclease  removes  nucleo-des  on  
the  new  strand  between  GATC  &  
mismatch  
• DNA  pol  replaces  the  nucleo-des,  
26  
correc-ng  the  mismatch   Animation
The  Problem  of  Strand  Discrimina-on  
• MMR  can  only  aid  if  repair  is  targeted  to  newly  synthesized  
strand  

• In  E.  coli,  this  is  accomplished  by  the  temporary  lack  of  
methyla-on  of  adenines  in  GA*TC  by  Dam  methylase  

• MutH  endonuclease  cleaves  only  unmethylated  GATC  sites,  

allowing  entry  on  newly  synthesized  strand  

• Dam  mutants  show  random  repair  of  either  DNA  strand  

27  
 3. Very  Short  Patch  Repair  (VSP)  
• Specialized  in  recognizing  T-­‐G  mismatch,  specifically  in    
– 5’  CmCWG  G  3’  
– 3’    G  GWCmC  5’  
– m  =  methyla-on;  W  =  either  A/T  or  T/A  

• DNA  cytosine  methylase  encoded  by  dcm  gene,  methylates  

the  2nd  cytosine  in  this  sequence    
• T-­‐G  mismatch  arise  upon  deamina-on  of  5-­‐methylcytosine  
to  thymine  
– thymine  at  a  posi-on  where  cytosine  should  be   28  
 VSP  –  3  Step  Process  
• Vsr  endonuclease,  encoded  by  vsr  
gene,  iden-fies  and  binds  to  T-­‐G  
mismatch  in  the  sequence  

• Bound  Vsr  catalyzes  a  break  in  the  

phosphodiester  bond  next  to  the  T  
comprising  the  mismatch  

• Exonuclease  ac-vity  removes  T  

and  polymeriza-on  ac-vity  adds  
the  correct  C  
29  
 Some  Thoughts  
• How  else  T-­‐G  mismatch  in  5’  CmCW  G  G  3’/3’  G  GWCmC  5’  
can  be  repaired?  
–  MutHLS  methyl  directed  mismatch  repair  
• Deamina-on  of  5-­‐methylcytosine  to  thymine  can  be  
independent  of  replica-on  
– DNA  may  not  be  in  hemi-­‐methylated  state  
– MutHLS  may  not  know  which  strand  to  target  
• Vsr  endonuclease  targets  thymine  arose  due  to  deamina-on  
• VSP  has  only  been  iden-fied  in  E.coli  
• However,  5-­‐methylcytosine  has  been  found  in  other  
organisms   30  
 4. Base  Excision  Repair  by  
Glycosylases  
• In    base  excision  repair  (BER),  a  modified  base  is  first  excised  
and  and  then  the  en-re  nucleo-de  is  replaced  
• E.coli  contain  many  types  of  glycosylases  
– class  of  repair  enzymes  that  excise  by  hydrolyzing  the  
glycosidic  linkage  between  a  base  and  its  deoxyribose  sugar  
leaving  an  AP  (apurinic  or  apyrimidinic)  site  

31  
Base  Excision  Repair  (BER)  
a) Each  DNA  glycosylase  recognizes  and  
removes  a  specific  type  of  damaged  
base,  producing  an  AP  site  

b) AP  endonuclease  cleaves  the  

phosphodiester  bond  on  the  5’  side  of  
AP  site  

c) Removes  the  deoxyribose  sugar  

d) DNA  polymerase  adds  new  nucleo-des  

to  the  exposed  3’  OH  group  

e) The  gap  in  sugar-­‐phosphate  backbone  is  

sealed  by  DNA  ligase,  restoring  original  
sequence  
32  
 e.g.  Uracil-­‐N-­‐glycosylase    
• Encoded  by  ung  gene  
• In  RNA  à  U  =  A  
• In  DNA  due  to  incorpora-on  errors  (A  =  U)  
or  due  to  deamina-on  of  cytosine  (U  =  G)  
 

• Addi-onal  glycosylases  repair  

deaminated,  alkylated,  oxidized  bases  
and  pyrimidine  dimers  
 
33  
Animation
 Double  Strand  Breaks  
• A  poten-ally  dangerous  type  of  DNA  damage  occurs  when  both  
strands  of  the  double  helix  are  broken  
– leaving  no  intact  template  strand  for  repair  
• Breaks  of  this  type  are  caused  by  ionizing  radia-on,  oxidizing  
agents  and  replica-on  errors  
• If  these  lesions  were  lec  unrepaired,  they  would  quickly  lead  to  
the  breakdown  of  chromosomes  into  smaller  fragments  
• Two  dis-nct  mechanisms  have  evolved  to  avoid  the  poten-al  
damage    
1. nonhomologous  end-­‐joining  
2. homologous  end-­‐joining  

34  
 Nonhomologous  end-­‐joining  
• The  broken  ends  are  juxtaposed  and  rejoined  by  DNA  
liga-on  
• Generally  with  the  loss  of  one  or  more  nucleo-des  at  the  
site  of  joining  
• An  emergency  solu-on  to  the  repair  of  double-­‐strand  breaks  
• A  common  outcome  in  mammalian  cells  
• Although  a  change  in  the  DNA  sequence  results  at  the  site  of  
breakage,  apparently  an  acceptable  solu-on  to  the  problem  
of  keeping  chromosomes  intact  

35  
 Homologous  end-­‐joining  
• Applicable  to  diploid  cells  
– two  copies  of  each  double  helix  
• Transfer  nucleo-de  sequence  informa-on  from  the  intact  
DNA  double  helix  to  the  site  of  the  double-­‐strand  break  in  
the  broken  helix  
• A  DNA  replica-on  process  then  uses  the  undamaged  
chromosome  as  the  template  for  transferring  gene-c  
informa-on  to  the  broken  chromosome,  repairing  it  with  no  
change  in  the  DNA  sequence  

36  
Two  different  types  of  end-­‐joining  
for  repairing  double-­‐strand  breaks  

37  
Mechanisms  that  Tolerate  DNA  Damage  
• What  happens  if  lesions  in  DNA  are  not  repaired  before  
replica-on  machinery  arempts  to  polymerize  across  a  
template?  
• Cross  linked  or  chemically  modified  bases  may  not  allow  to  
move  through  the  lesion  containing  area  
• By  using  “tolera-ng  mechanisms”,  replica-on  machinery  
can  proceed  through  certain  types  of  lesions  
• They  do  not  repair  the  lesions  
– only  provide  the  means  to  get  the  DNA  replicated  
• Two  mechanisms  that  tolerate  pyrimidine  dimers  
1. Transdimer  synthesis  
2. Post  replica-on/  recombina-on  (PRR)   38  
 1. Transdimer  Synthesis  
• Also  known  as  SOS  mutagenic  repair  (last  arempt  to  save)  

• During  replica-on  of  UV  damaged  DNA  

– DNA  pol  III  encounters  dimer  lesion  

– Any  nucleo-de  arempted  to  pair  will  not  pair  

– β  subunit  of  DNA  pol  does  the  edi-ng/  proofreading  func-on  

• 2  subunits  form  a  circular  clamp  that  anchors  DNA  pol  to  DNA  

• β  clamp  will  not  allow  DNA  pol  to  add  the  next  nucleo-de  if  
there  are  distor-ons  

39  
 DNA  polymerase  III  

Source: http://www.ebi.ac.uk/pdbe/quips?story=BetaClamp 40  
1. Transdimer  Synthesis  Cont.  
– 2  gene  products,  UmuD  and  
umuC,  from  SOS  regulon  
subs-tutes  for  β  clamp  à  
relaxes  the  clamp  

– This  relaxa-on  allows  

polymeriza-on  to  con-nue  
across  lesions  

– highly  error  prone  à  

higher  rate  of  mutagenesis  
in  UV  damaged  cells  

41  
 2. Post  Replica-on/  Recombina-on  
• Homologous  recombina-on  is  another  mechanism  used  in  
tolera-ng  lesions  
– break  nearly  iden-cal  DNA  molecules  and  rejoin  in  different  
combina-ons  
– enzyme  involved  is  mul-func-onal  RecA  
• binds  to  ss  DNA  
• promotes  ss  DNA  base  pairing  with  homologous  sequence  
(similar,  not  iden-cal)  from  a  ds  DNA    
• Rec  A  unwinds  dsDNA  
– another  enzyme  involved  is  RecBCD  (aka  Exonuclease  V)  
• mul-func-onal  –  exonuclease  for  ds  DNA,  helicase  for  ds  DNA,  
both  exo/endo  nuclease  for  ss  DNA  
42  
 Post  Replica-on/  Recombina-on  (PRR)  
• PRR  begins  with  post  dimer  ini-a-on  
– occurs  when  DNA  pol  III  arempts  to  replicate  through  a  lesion  
– DNA  pol  III  temporarily  stalls  upon  the  lesion  
– it  then  commences  polymeriza-on  some  distance  away  from  
lesion  (skips  over  upto  800  bp)  
– a  gap  (opposite  to  dimer  in  the  template)  is  lec  in  the  new  
daughter  strand  –  what  happens  if  the  gap  is  not  filled?  
– both  strands  now  contain  a  damage  
• Gap  filling  by  recombina-on    
– occurs  post  replica-on  à  PRR  
– PRR  is  a  6  step  process  
43  
 PRR  Process  

44  
 PRR  
1. Gap  forms  in  the  new  daughter  strand;  RecA  binds  to  the  ss  
region  of  the  gap  
2. RecBCD  cuts  the  undamaged  parent  strand  
3. RecA  coated  gap  strand  aligns  and  H  bonds  (assimilates)  
with  the  complementary  region  of  the  undamaged  parent  
strand  (not  the  template)  
4. An  endonuclease  frees  the  parent  strand  with  another  cut  
5. Ligase  seals  the  gaps  in  the  damaged  ds  DNA  
6. DNA  pol  I  fills  in  the  gap  in  the  parental  strand  

45  
 PRR  Process  

46  
 Summary  In-­‐Class  &  Online  
1. Mechanisms  that  Reverse  DNA  Damage  
– photoreac-va-on  
– methyltransferase  
2. Mechanisms  that  Excise  DNA  Damage  
– Nucleo-de  excision  repair  (NER)  
– Mismatch  repair  
– Very  short  patch  (VSP)  repair  
– Glycosylases  
3. Mechanisms  that  Tolerate  DNA  Damage  
– Transdimer  synthesis  
– Post  replica-on/  recombina-on  (PRR)   47  
 References  
• Ch.  10  from  Modern  Gene-c  Analysis  
• Chapter  18  from  Gene-cs:  A  Conceptual  Approach,  3rd  edi-on  ©  2008  
by  B.A.  Pierce,  New  York:  W.  H.  Freeman  
• Chapter  4  from  Fundamental  Bacterial  Gene-cs  by  N.  Trun  and  J.  
Trempy.  2004.  Blackwell  Publishing  
• Chapter  15  from  Concepts  of  Gene-cs  by  W.S.  Klug,  M.R.  Cummings,  
C.A.  Spencer  and  M.A.  Palladino.  10th  edi-on.  2012.  Pearson  Educa-on  
Inc.    

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