1. Chapter 20
Repair Systems

2. Injury to DNA is minimized by systems that recognize and correct the
20.1 Introduction
Injury to DNA is minimized by systems that recognize and correct the
damage. The repair systems are as complex as the replication
apparatus itself, which indicates their importance for the survival of
the cell.
The importance of DNA repair in eukaryotes is indicated by the
identification of >130 repair genes in the human genome.
We may divide the repair systems into several general types, as
summarized in Figure 20.1.
Mismatches are usually corrected by excision repair (base excision
repair and nucleotide excision repair).
Figure: 9.1
Title:
One strand of DNA is transcribed into RNA
Caption:
The function of RNA polymerase is to copy one strand of duplex DNA into RNA.

3. Figure Repair genes can be classified into pathways that use different mechanisms to reverse or bypass damage to DNA.
Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.

4. Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.
Figure Excision repair directly replaces damaged DNA and then resynthesizes a replacement stretch for the damaged strand.

5. 20.2 Repair Systems Correct Damage to DNA
Key Concepts
Repair systems recognize DNA sequences that do not conform to standard
base pairs.
Excision systems remove one strand of DNA at the site of damage and then
replace it.
Recombination-repair systems use recombination to replace the double-
stranded region that has been damaged.
All these systems are prone to introducing errors during the repair process.
Photoreactivation is a nonmutagenic repair system that acts specifically on
pyrimidine dimers.
Figure: 9.46
Title:
Bacterial termination occurs at a discrete site
Caption:
The DNA sequences required for termination are located prior to the terminator sequence. Formation of a hairpin in the RNA may be necessary.
The types of damage that trigger repair systems can be divided into two general classes: single-base changes and structural distortions.
pyrimidine dimer : covalent bonds between two adjacent pyrimidine bases that are introduced by ultraviolet (UV) irradiation.

6. Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.
Figure Deamination of cytosine creates a U-G base pair. Uracil is preferentially removed from the mismatched pair.

7. Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.
Figure A replication error creates a mismatched pair that may be corrected by replacing one base; if uncorrected, a mutation is fixed in one daughter duplex.

8. Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.
Figure Ultraviolet irradiation causes dimer formation between adjacent thymines. The dimer blocks replication and transcription.

9. Figure: 9.32
Title:
Sigma controls promoter recognition
Caption:
The sigma factor associated with core enzyme determines the set of promoters where transcription is initiated.
Figure Methylation of a base distorts the double helix and causes mispairing at replication. Star indicates the methyl group.

10. Figure: 9.32
Title:
Sigma controls promoter recognition
Caption:
The sigma factor associated with core enzyme determines the set of promoters where transcription is initiated.
Figure Depurination removes a base from DNA, blocking replication and transcription.

11. 20.3 Excision Repair Systems in E. coli
Key Concepts
The Uvr system makes incisions ~12 bases apart on both sides of damaged
DNA, removes the DNA between them, and resynthesizes new DNA.
Excision repair systems vary in their specificity, but share the same
general features. Each system removes mispaired or damaged
bases from DNA and then synthesizes a new stretch of DNA to
replace them.
Figure: 9.46
Title:
Bacterial termination occurs at a discrete site
Caption:
The DNA sequences required for termination are located prior to the terminator sequence. Formation of a hairpin in the RNA may be necessary.
incision : an endonuclease recognizes the damaged area in the
DNA, and isolates it by cutting the DNA strand on both sides of the
damage.
excision : a 5’-3’ exonuclease removes a stretch of the damaged
strand.

12. Figure 20.9 Excision-repair removes and replaces a stretch of DNA that includes the damaged base(s).
Title:
Sigmas control phage development
Caption:
Transcription of phage SPO1 genes is controlled by two successive substitutions of the sigma factor that change the initiation specificity.

13. Figure The Uvr system operates in stages in which UvrAB recognizes damage, UvrBC nicks the DNA, and UvrD unwinds the marked region. (The uvr system of excision repair includes three genes, uvrA, B, and C, which code for the components of a repair endonuclease. UvrD is a helicase. In almost all (99%) of cases, the average length of replaced DNA is ∼12 nucleotides (short-patch repair).
Figure: 9.41
Title:
Sigmas control phage development
Caption:
Transcription of phage SPO1 genes is controlled by two successive substitutions of the sigma factor that change the initiation specificity.

14. 20.4 Excision-Repair Pathways in Mammalian Cells
Key Concepts
Mammalian excision repair is triggered by directly removing a damaged base
from DNA.
Base removal triggers the removal and replacement of a stretch of
polynucleotides.
The nature of the base removal reaction determines which of two pathways
for excision repair is activated.
The polδ/ε pathway replaces a long polynucleotide stretch; the polβ pathway
replaces a short stretch.
The general principle of excision-repair in mammalian cells is similar
to that of bacteria. The process usually starts in a different way,
however, with the removal of an individual damaged base.
Enzymes that remove bases from DNA are called glycosylases and
lyases.
Figure: 9.46
Title:
Bacterial termination occurs at a discrete site
Caption:
The DNA sequences required for termination are located prior to the terminator sequence. Formation of a hairpin in the RNA may be necessary.

15. Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.
Figure A glycosylase removes a base from DNA by cleaving the bond to the deoxyribose.

16. Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.
Figure A glycosylase hydrolyzes the bond between base and deoxyribose (using H2O), but a lyase takes the reaction further by opening the sugar ring (using NH2).

17. Figure Base removal by glycosylase or lyase action triggers mammalian excision-repair pathways.
Figure: 9.30
Title:
RNA polymerase contacts one face of DNA
Caption:
One face of the promoter contains the contact points for RNA.

18. 20.5 Base Flipping Is Used by Methylases and Glycosylases
Key Concepts
Uracil and alkylated bases are recognized by glycosylases and removed
directly from DNA.
Pyrimidine dimers are reversed by breaking the covalent bonds between
them.
Methylases add a methyl group to cytosine.
All these types of enzyme act by flipping the base out of the double helix
where, depending on the reaction, it is either removed or is modified and
returned to the helix.
Figure: 9.48
Title:
A rho-dependent terminator has a biased base composition
Caption:
A rho-dependent terminator has a sequence rich in C and poor in G preceding the actual site(s) of termination. The sequence is shown in the form of the RNA. It represents the 3' end of the RNA.

19. Several enzymes that remove or modify individual bases in DNA use
a remarkable reaction in which a base is “flipped” out of the double
helix.
One of the most common reactions in which a base is directly
removed from DNA is catalyzed by uracil-DNA glycosylase. Uracil
typically occurs in DNA because of a (spontaneous) deamination of
cytosine. It is recognized by the glycosylase and removed. The
reaction is similar to that of the methylase.
Alkylated bases (typically in which a methyl group has been added to
a base) are removed by a similar mechanism.
Another enzyme to use base flipping is the photolyase that reverses
the bonds between pyrimidine dimers (see Figure 20.5).
Figure: 9.1
Title:
One strand of DNA is transcribed into RNA
Caption:
The function of RNA polymerase is to copy one strand of duplex DNA into RNA.

20. Figure: 9.43
Title:
Sigmas change in both compartments during sporulation
Caption:
Sporulation involves successive changes in the sigma factors that control the initiation specificity of RNA polymerase. The cascades in the forespore (left) and the mother cell (right) are related by signals passed across the septum (indicated by horizontal arrows).
Figure A methylase "flips" the target cytosine out of the double helix in order to modify it.

21. 20.6 Error-Prone Repair and Mutator Phenotypes
Key Concepts
Damaged DNA that has not been repaired causes DNA polymerase III to stall
during replication.
DNA polymerase V (coded by umuCD), or DNA polymerase IV (coded by
dinB) can synthesize a complement to the damaged strand.
The DNA synthesized by the repair DNA polymerase often has errors in its
sequence.
Proteins that affect the fidelity of replication may be identified by mutator
genes, in which mutation causes an increased rate of spontaneous mutation.
Figure: 9.48
Title:
A rho-dependent terminator has a biased base composition
Caption:
A rho-dependent terminator has a sequence rich in C and poor in G preceding the actual site(s) of termination. The sequence is shown in the form of the RNA. It represents the 3' end of the RNA.
Error-prone : occurs when DNA incorporates noncomplementary
bases into the daughter strand.

22. 20.7 Controlling the Direction of Mismatch Repair
Key Concepts
The mut genes code for a mismatch-repair system that deals with
mismatched base pairs.
There is a bias in the selection of which strand to replace at mismatches.
The strand lacking methylation at a hemimethylated GATC/CTAG is usually
replaced.
This repair system is used to remove errors in a newly synthesized strand of
DNA. At G-T and C-T mismatches, the T is preferentially removed.
mutator : a gene whose mutation results in an increase in the basal level of mutation of the genome. Such genes are often code for proteins that are involved in repairing damaged DNA.
Figure: 9.53
Title:
Antiterminators can act at different locations in the transcription unit
Caption:
Host RNA polymerase transcribes lambda genes and terminates at t sites. pN allows it to read through terminators in the L and R1 units; pQ allows it to read through the R' terminator. The sites at which pN acts (nut) and at which pQ acts (qut) are located at different relative positions in the transcription units.

23. Figure: 9.44
Title:
The forespore communicates with the mother
Caption:
sF triggers synthesis of the next sigma factor in the forespore (sG) and turns on SpoIIR which causes SpoIIGA to cleave pro-sE.
Figure Preferential removal of bases in pairs that have oxidized guanine is designed to minimize mutations.

24. Figure GATC sequences are targets for the Dam methylase after replication. During the period before this methylation occurs, the nonmethylated strand is the target for repair of mismatched bases.
Figure: 9.45
Title:
Several pathways cross the septum
Caption:
The crisscross regulation of sporulation coordinates timing of events in the mother cell and forespore.

25. Figure MutS recognizes a mismatch and translocates to a GATC site. MutH cleaves the unmethylated strand at the GATC. Endonucleases degrade the strand from the GATC to the mismatch site.
Figure: 9.46
Title:
Bacterial termination occurs at a discrete site
Caption:
The DNA sequences required for termination are located prior to the terminator sequence. Formation of a hairpin in the RNA may be necessary.

26. Figure 20.17 The MutS/MutL system initiates repair of mismatches produced by replication slippage.
Title:
An intrinsic terminator has two features
Caption:
Intrinsic terminators include palindromic regions that form hairpins varying in length from 7-20 bp. The stem-loop structure includes a G-C-rich region and is followed by a run of U residues.

27. 20.8 Recombination-Repair Systems in E. coli
Key Concepts
The rec genes of E. coli code for the principal retrieval system.
The principal retrieval system functions when replication leaves a gap in a
newly synthesized strand that is opposite a damaged sequence.
The single strand of another duplex is used to replace the gap.
The damaged sequence is the removed and resynthesized.
Recombination-repair systems use activities that overlap with those
involved in genetic recombination. They are also sometimes called
“post-replication repair.” because they function after replication. Such
systems are effective in dealing with the defects produced in
daughter duplexes by replication of a template that contains
damaged bases.
Figure: 9.55
Title:
Termination and antitermination factors bind to RNA polymerase
Caption:
Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.
single-strand exchange : the gap opposite the damaged site in the
first duplex is filled by stealing the homologous single strand of DNA
from the normal duplex.

28. Figure An E. coli retrieval system uses a normal strand of DNA to replace the gap left in a newly synthesized strand opposite a site of unrepaired damage.
Figure: 9.49
Title:
Rho terminates transcription
Caption:
Rho factor pursues RNA polymerase along the RNA and can cause termination when it catches the enzyme pausing at a rho-dependent terminator.

29. be converted to a duplex and DSB by resolvases.
20.9 Recombination Is an Important Mechanism to Recover from Replication Errors
Key Concepts
A replication fork may stall when it encounters a damaged site or a nick in
DNA.
A stalled fork may reverse by pairing between the two newly synthesized
strands.
A stalled fork may restart repairing the damage and using a helicase to move
the fork forward.
The structure of the stalled fork is the same as a Holliday junction and may
be converted to a duplex and DSB by resolvases.
All cells have many pathways to repair damage in DNA. Excision-
repair pathways can in principle be used at any time, but
recombination-repair can be used only when there is a second
duplex with a copy of the damaged sequence, that is, postreplication.
Recombination-repair pathways are involved in allowing the fork to
be restored after the damage has been repaired or to allow it to
bypass the damage.
Figure: 9.55
Title:
Termination and antitermination factors bind to RNA polymerase
Caption:
Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.

30. Figure A replication fork stalls when it reaches a damaged site in DNA. Reversing the fork allows the two daughter strands to repair. After the damage has been repaired, the fork is restored by forward-branch migration catalyzed by a helicase.
Figure: 9.50
Title:
Rho can terminate when a nonsense mutation removes ribosomes
Caption:
The action of rho factor may create a link between transcription and translation when a rho-dependent terminator lies soon after a nonsense mutation.

31. Figure The structure of a stalled replication fork resembles a Holliday junction and can be resolved in the same way by resolvases. The results depend on whether the site of damage contains a nick. Result 1 shows that a double-strand break is generated by cutting a pair of strands at the junction. Result 2 shows a second DSB is generated at the site of damage if it contains a nick. Arrowheads indicate 3′ ends.
Figure: 9.50
Title:
Rho can terminate when a nonsense mutation removes ribosomes
Caption:
The action of rho factor may create a link between transcription and translation when a rho-dependent terminator lies soon after a nonsense mutation.

32. Figure When a replication fork stalls, recombination-repair can place an undamaged strand opposite the damaged site. This allows replication to continue.
Figure: 9.51
Title:
Action at a terminator controls transcription
Caption:
Antitermination can be used to control transcription by determining whether RNA polymerase terminates or reads through a particular terminator into the following region.

33. 20.10 RecA Triggers the SOS System
Key Concepts
Damage to DNA causes RecA to trigger the SOS response, which consists of
genes coding for many repair enzymes.
RecA activates the autocleavage activity of LexA.
LexA represses the SOS system; its autocleavage activates those genes.
SOS response : the coordinate induction of many genes whose
products include repair functions, in response to irradiation or other
damage to DNA; results from activation of protease activity by RecA
to cleave LexA repressor.
SOS box : the DNA sequence (operator) of ~20 bp recognized by
LexA repressor protein.  
Figure: 9.55
Title:
Termination and antitermination factors bind to RNA polymerase
Caption:
Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.

34. Figure: 9.54
Title:
Termination is prevented by factors that act at nut
Caption:
Ancillary factors bind to RNA polymerase as it passes the nut site. They prevent rho from causing termination when the polymerase reaches the terminator.
Figure The LexA protein represses many genes, including repair functions, recA and lexA. Activation of RecA leads to proteolytic cleavage of LexA and induces all of these genes.

35. 20.11 Eukaryotic Cells Have Conserved Repair Systems
Key Concepts
The yeast RAD mutations, identified by radiation sensitive phenotypes, are in
genes that code for repair systems.
Xeroderma pigmentosum (XP) is a human disease caused by mutations in
any one of several repair genes.
A complex of proteins including XP products and the transcription factor THIIH
provides a human excision-repair mechanism.
Transcriptionally active genes are preferentially repaired.
The types of repair functions recognized in E. coli are common to a
wide range of organisms. The best characterized eukaryotic systems
are in yeast, where Rad51 is the counterpart to RecA.
In yeast, the main function of the strand-transfer protein is
homologous recombination.
Many of the repair systems found in yeast have direct counterparts in
higher eukaryotic cells, and in several cases these systems are
involved with human diseases.
Figure: 9.55
Title:
Termination and antitermination factors bind to RNA polymerase
Caption:
Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.

36. Figure A helicase unwinds DNA at a damaged site, endonucleases cut on either side of the lesion, and new DNA is synthesized to replace the excised stretch.
Figure: 9.55
Title:
Termination and antitermination factors bind to RNA polymerase
Caption:
Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.

37. 20.12 A Common System Repairs Double-Strand Breaks
Key Concepts
The NHEJ pathway can ligate blunt ends of duplex DNA.
Mutations in the NHEJ pathway cause human diseases.
non-homologous end-joining (NHEJ) ligates blunt ends. It is the
major mechanism to repair the double-strand breaks.
Figure: 9.55
Title:
Termination and antitermination factors bind to RNA polymerase
Caption:
Ancillary factors bind to RNA polymerase as it passes certain sites. The nut site consists of two sequences. NusB-S10 join core enzyme as it passes boxA. Then NusA and pN protein bind as polymerase passes boxB. The presence of pN allows the enzyme to read through the terminator, producing a joint mRNA that contains immediate early sequences joined to delayed early sequences.

38. Figure: 9.54
Title:
Termination is prevented by factors that act at nut
Caption:
Ancillary factors bind to RNA polymerase as it passes the nut site. They prevent rho from causing termination when the polymerase reaches the terminator.
Figure Nonhomologous end joining requires recognition of the broken ends, trimming of overhanging ends and/or filling, followed by ligation.

39. Figure The Ku70-Ku80 heterodimer binds along two turns of the DNA double helix and surrounds the helix at the center of the binding site
Figure: 9.54
Title:
Termination is prevented by factors that act at nut
Caption:
Ancillary factors bind to RNA polymerase as it passes the nut site. They prevent rho from causing termination when the polymerase reaches the terminator.

40. Figure: 9.54
Title:
Termination is prevented by factors that act at nut
Caption:
Ancillary factors bind to RNA polymerase as it passes the nut site. They prevent rho from causing termination when the polymerase reaches the terminator.
Figure If two heterodimers of Ku bind to DNA, the distance between the two bridges that encircle DNA is ~12 bp.