Lecture 10

DNA REPAIR CONTINUED

Types of mutations

Deletions a part of the DNA is missing anywhere from 1 base pair to parts of chromosomes.

Insertions of new DNA again ranging from 1 to many base pairs

Point mutations; a change in the nucleotide. Two types

Transitions Purine to other purine or pyrimidine to other pyrimidine.

Transversions: Purine to Pyrimidine or Pyrimidine to Purine.

"5’----------GAATTC---------3’"

5’----------GAATTC---------3’

3’----------CTTAAG---------5’

Substitutions:

5’----------GATTTC---------3’

3’----------CTAAAG---------5’

5’----------GAGTTC---------3’

3’----------CTCAAG---------5’

Deletion:

5’----------GAATC----------3’

3’----------CTTAG----------5’

"5’----------GAATTC---------3’"

5’----------GAATTC---------3’

3’----------CTTAAG---------5’

Insertion:

5’----------GAACTTC---------3’

3’----------CTTGAAG---------5’

Duplication:

5’----------GAATATTC--------3’

3’----------CTTATAAG--------5’

Substitutions that occur in protein-coding sequences

Silent- changes a codon, but not the encoded amino acid residue

possible because the code is degenerate

Missense- changes the encoded residue

Nonsense- an amino acid-encoding codon becomes a stop codon

EXAMPLES - substitutions

Silent- TGT (Cys)--> TGC (Cys)

GCA (Ala)--> GCN (Ala)

(N = any)

Missense- TGT (Cys)--> TGG (Trp)

Nonsense- TGT (Cys)--> TGA (STOP)

Frameshift example

NH2- Met-Thr-Leu -Lys -COOH

5’-ATG-ACC-TTG-AAA-TAA-3’

NH2-Met-Pro -COOH

5’-ATG-CCT-TGA-AAT-AA-3’

SNPs

SNPs can have variable effects

SNPs can have no effect. Their change can be neutral to the protein, e.g. a silent mutation.

SNPs can have a subtle effect, e.g lys to arg (both are polar basic). This is what we suspect is happening in complex genetic diseases

SNPs can have measurable effects (a pronounced reduction in activity).

SNPs can change protein function. A new substrate might be recognized.

SNPs can complete eliminate the proteins ability to function.

Fate of DNA damage

Tolerated (ignored)

Repaired

Can kill the cell or cause the cell to kill itself

Can become fixed, resulting in a mutation

(Note: fixed <> repaired)

Examples of mutation fixation

Replication of an unrepaired misincorporation

Replication of an unrepaired cytosine deamination

(deaminated cytosine = uracil)

Human Genome

Haploid size = 3300 Megabase pairs

= 3.3 x 10⁹ (= billion) base pairs

Diploid size = double that

Misincorporation (10^-5) x not proofread (10^-2) x escape mismatch repair (10^-3) = 10^-10

Thus, less than one replication error is fixed per cell division

Mutation Rate per bp

10^-9 per base pair per cell division

This refers to mutations that are not repaired (i.e. they’re fixed)

Thus, there are at least six new base changes in each kid that were not present in either parent, but this is an underestimate as there’s more since they accumulate in the germ line stem cells as the father ages

Remember, most of these are not in genes

Mutation rate per gene

From all sources (misincorps, damage):

Approx 10^-5 per gene per cell division

Human genome contains 30,000-100,000 genes

Thus, roughly one new mutation (allele) is created per cell division (most likely recessive)

DNA mismatch repair

MMR cont

Uracil DNA glycosylase and BER

An enzyme that removes Uracil from DNA

Resulting abasic site is filled in by polymerase

Uracil in DNA comes mainly from deamination of cytosine

That may be why DNA uses thymine instead of uracil

If the uracil isn’t removed, it will pair with A, causing C/G --> T/A transition.

BER

Environmental DNA damage

The discovery of NER

Setlow found three mutations in E. coli that rendered the cells sensitive to UV damage.

The genes were named UvrA, UvrB and UvrC for UV resistance.

Using cell-free extracts, Sancar determined the mechanism of uvrABC which has been refined over the years.

The process of uvrABC excision of DNA damage is called nucleotide excision repair.

NER in E. coli continued

NER in mammalian cells

A disease in humans known as Xeroderma Pigmentosum XP is a rare inherited disease of humans which, among other things, predisposes the patient to

pigmented lesions on areas of the skin exposed to the sun and

an elevated incidence of skin cancer.

It turns out that XP can be caused by mutations in any one of several genes - all of which have roles to play in NER.

James Cleaver went around and collected cells from hundreds of these patients. He then figured out that the disease was made up of eight genes named XP-A through XP-G plus one called XP-V for variant.

Slide 27

There are 8 XP complementation groups

XP-A participates in photoproduct recognition and DNA binding This binding may be followed by the formation of a quasi-stable complex consisting of XPA, XPC, human single-strand binding protein (RPA/HSSB), and TFIIH, which then acts as a nucleation site for binding of the incision/excision enzymes.

XP-B is a 3’-> 5’ DNA helicase that may be involved in unwinding the DNA 5'-ward of a damaged base

XP-C is a single-stranded DNA binding protein that is essential for repair of the nontranscribed regions of the genome, that acts in the initial step of damage recognition.

XP-D is a 5'–3' helicase, a component of transcription factor TFIIH may be involved in 3'-ward unwinding of the DNA in the vicinity of a damaged base

XB Genes continued

XP-E is thought to be involved with the recognition of damaged DNA because it has the capacity to bind to UV-damaged DNA

XP-F in association with the ERCC1 protein, incises DNA on the 5' side of the damaged site

XP-G incises DNA 3' to the damaged site

XP-V protein is a low-fidelity class Y DNA polymerase, that can replicate UV-induced pyrimidine dimers in vivo with the insertion of the correct bases in the daughter strand

CSA likely participates in a CSB/RNA polII complex stalled at damaged sites in transcriptionally active DNA that helps remove the stalled RNA polII from the DNA damage site.

CSB is believed to be a DNA helicase that is required for ubiquitinating RNA polII for its remove and degradation at sites of DNA damage.

Some XP proteins are

XPA, which encodes a protein that binds the damaged site and helps assemble the other proteins needed for NER.

XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD also produce signs of premature aging.

XPF, with ERCC1 cuts the backbone on the 5' side of the damage

XPG, which cuts the backbone on the 3' side.

XPC interacts with HR23B in GGR and recognizes damage

XPD is a DNA helicase in in transcription complex

VPV is a by-pass polymerase

Cleaver’s study: Complementation groups of XP

Developing an in vitro DNA repair system

The in vitro assay

How the incision product is detected

Slide 35

Slide 36

Global Genomic Repair

Human global genome NER. (a) In the damage recognition step, the XPC-hHR23B complex recognizes the damage (a pyrimidine dimer in this case), binds to it, and causes localized DNA melting. XPA also aids this process. RPA binds to the undamaged DNA strand across from the damage. (b) The DNA helicase activity of TFIIH causes increased DNA melting. (c) RPA helps position two endonucleases (the ERCC1-XPF complex and XPG) on either side of the damage, and these endonucleases clip the DNA. (d) With the damaged DNA removed on a fragment 24-32 nt long, DNA polymerase fills in the gap with good DNA and DNA ligase seals the final nick.

There are endogenous and exogenous sources of mutagens

Mutagens are any reagent that causes changes in DNA (often referred to as DNA damage) that can ultimately lead to a change in the DNA sequence.

Examples of endogenous reagents are; free radicals generated during oxidation reactions, pH changes that can lead to changes in DNA, errors in DNA replication and recombination errors.

Examples or exogenous reagents are UV radiation, ionizing radiation, chemicals such as benzopyrene and and natural compounds such as aflatoxin.

What do mutagens do?

Mutagens primarily affect DNA by causing a physical change in the structure, which ultimately alters the sequence, leading to changes in genes such that the information is altered. This leads to loss of a protein, a change in the sequence (and likely structure) of a protein or a change in the level of proteins found in cells.

Types of Mutagens

A variety of chemicals react directly with DNA. Alkylating agents are electrophiles that add methyl, ethyl and more complicated alkyl groups to nucleic acid bases. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in vivo becomes a highly reactive methylating agent. Electrophilic reactants can also be generated by cytochrome P450 oxidation of xenochemicals. These chemicals include benzo[a]pyrene, acetylaminofluorene and aflatoxin. Bulky adducts result. Nitrogen and sulfur mustards (used in chemical warfare) link bases on opposite DNA strands, creating cross-links.

Alkylating agents

Reactive sites on nucleotides

Alkylating agents

Mutagens don’t always start out that way

Many compounds that enter out cells are lipophilic (typically organic compounds). These compounds are not reactive with DNA. A system of enzymes called P450 monooxygenases add oxygen molecules in order to make them more soluble but this also makes them reactive with DNA.

Cytochrome P450 monooxygenase system

Xenobiotics are chemical compounds that do not belong to the normal composition of the human body. These compounds enter the body via the diet, air and medication. The principal route of elimination of xenobiotics from the body is biotransformation. They are eliminated by microsomal phase I and microsomal and cytosolic phase II drugmetabolising enzymes. These enzymes add functional groups to make lipophilic molecules more hydrophilic and hence easier to eliminate. The oxidative reactions are mainly catalysed by cytochrome P450 (CYP or P450) enzymes. The CYP superfamily of microsomal hemoproteins catalyses the monooxygenation of a large number of endogenous and exogenous compounds. They play a key role in the

metabolism of a wide variety of xenobiotics, such as drugs, pesticides and (pre)carcinogens.

General reaction

Aflatoxin reaction

EMS modification of DNA

P450s are found in all cells but mostly in liver and small intestine

P450 at work

Slide 51

Endogenous DNA damage

Replication errors

Polymerase misincorporation

Recombination errors

Unequal crossing over, etc.

Spontaneous base damage

Deaminations, depurinations

Byproducts of metabolism

Oxygen radicals

Deamination of Cytosine

Sources of exogenous DNA damage

Chemicals

Natural

In foods, e.g. aflatoxin

Man-made/man-increased

Nitrogen Mustard - WWI nerve gas

Benzopyrene - smoke from coal, autos, cigs

Ultraviolet (UV) Radiation (from sun)

Ionizing radiation

Natural: radon gas, cosmic rays

Man-made: x-rays, nuclear tests

UV Radiation,
Pyrimidine dimers

5’--CCGAATTCAG--3’

3’--GGCTTAAGTC--5’

How does DNA
damage cause mutations?

In many ways

Slide 57

Slide 58

See the man pat the pet cat

See tem anp att hep etc at

Examples of repair mech’s

Polymerase proofreading

DNA mismatch repair

Uracil DNA glycosylase

Nucleotide excision repair

DNA polymerases

Are proteins that replicate DNA

Have multiple domains or subunits

A good polymerase domain has a misincorporation rate of 10^-5(1/100,000)

Any misincorps are clipped off with 99% efficiency by the “proofreading” activity of the polymerase

Slide 62

Nucleotide Excision Repair

Carried out by a multi-protein complex

Removes bulky adducts from DNA, e.g.

Pyrimidine dimers caused by UV

Benzopyrene-DNA adducts

Nearby nucleotides are also excised

Resulting single-strand gap is filled in by polymerase

Nucleotide Excision Repair

5’--------CCGAAttCAG-------3’

3’--------GGCTTAAGTC-------5’

"5’--------CCGAAttCAG-------3’"

5’--------CCGAAttCAG-------3’

3’--------GGCTTAAGTC-------5’

INCISION

5’------- CCGAAttCAG ------3’

3’--------GGCTTAAGTC-------5’

EXCISION

5’-------- -------3’

3’--------GGCTTAAGTC-------5’

GAP -FILLING (BY A POLYMERASE)

5’--------CCGAATTCAG-------3’

3’--------GGCTTAAGTC-------5’

Slide 66

DNA Repair part 2

Repair of other DNA damage

Slide 68

Slide 69

Slide 70

Slide 71

Types of UV damage

Slide 73

Slide 74

Slide 75

Slide 76

Slide 77

Nucleotide Excision Repair

How UVDE works

Mismatch repair

DNA repair diseases

Xeroderma Pigmentosum

Autosomal recessive, multigenic, very rare

Symptoms:

Dry scaly skin (xeroderma)

Freckling; pigmentation abnormalities (pigmentosum)

Extreme sensitivity to sunlight

Greatly increased incidence of skin cancer (1000 X)

Neurological abnormalities

Defect in nucleotide excision repair

Xeroderma Pigmentosum

DNA repair diseases

HNPCC

Heriditary nonpolyposis colorectcal cancer

Autosomal dominant, multigenic, up to 1/200

Symptoms:

High frequency of colon and several other cancers

Defect in mismatch repair

Slide 84


	Deletions a part of the DNA is missing anywhere from 1 base pair to parts of chromosomes.
	Insertions of new DNA again ranging from 1 to many base pairs
	Point mutations; a change in the nucleotide. Two types
		Transitions Purine to other purine or pyrimidine to other pyrimidine.
		Transversions: Purine to Pyrimidine or Pyrimidine to Purine.


	5’----------GAATTC---------3’
	3’----------CTTAAG---------5’
	Substitutions:
	5’----------GATTTC---------3’
	3’----------CTAAAG---------5’
	5’----------GAGTTC---------3’
	3’----------CTCAAG---------5’
	Deletion:
	5’----------GAATC----------3’
	3’----------CTTAG----------5’


	5’----------GAATTC---------3’
	3’----------CTTAAG---------5’
	Insertion:
	5’----------GAACTTC---------3’
	3’----------CTTGAAG---------5’
	Duplication:
	5’----------GAATATTC--------3’
	3’----------CTTATAAG--------5’


	Silent- changes a codon, but not the encoded amino acid residue
		possible because the code is degenerate
	Missense- changes the encoded residue
	Nonsense- an amino acid-encoding codon becomes a stop codon


	Silent- TGT (Cys)--> TGC (Cys)
	GCA (Ala)--> GCN (Ala)
		(N = any)

	Missense- TGT (Cys)--> TGG (Trp)

	Nonsense- TGT (Cys)--> TGA (STOP)


	NH2- Met-Thr-Leu -Lys -COOH
	5’-ATG-ACC-TTG-AAA-TAA-3’



	NH2-Met-Pro -COOH
	5’-ATG-CCT-TGA-AAT-AA-3’


	SNPs can have no effect. Their change can be neutral to the protein, e.g. a silent mutation.
	SNPs can have a subtle effect, e.g lys to arg (both are polar basic). This is what we suspect is happening in complex genetic diseases
	SNPs can have measurable effects (a pronounced reduction in activity).
	SNPs can change protein function. A new substrate might be recognized.
	SNPs can complete eliminate the proteins ability to function.


	Tolerated (ignored)
	Repaired
	Can kill the cell or cause the cell to kill itself
	Can become fixed, resulting in a mutation
	(Note: fixed <> repaired)


	Replication of an unrepaired misincorporation
	Replication of an unrepaired cytosine deamination
	(deaminated cytosine = uracil)


	Haploid size = 3300 Megabase pairs
	= 3.3 x 10⁹ (= billion) base pairs
	Diploid size = double that
	Misincorporation (10^-5) x not proofread (10^-2) x escape mismatch repair (10^-3) = 10^-10
	Thus, less than one replication error is fixed per cell division



	10^-9 per base pair per cell division
	This refers to mutations that are not repaired (i.e. they’re fixed)
	Thus, there are at least six new base changes in each kid that were not present in either parent, but this is an underestimate as there’s more since they accumulate in the germ line stem cells as the father ages
	Remember, most of these are not in genes


	From all sources (misincorps, damage):
	Approx 10^-5 per gene per cell division
	Human genome contains 30,000-100,000 genes
	Thus, roughly one new mutation (allele) is created per cell division (most likely recessive)


	An enzyme that removes Uracil from DNA
	Resulting abasic site is filled in by polymerase
	Uracil in DNA comes mainly from deamination of cytosine
	That may be why DNA uses thymine instead of uracil
	If the uracil isn’t removed, it will pair with A, causing C/G --> T/A transition.


	Setlow found three mutations in E. coli that rendered the cells sensitive to UV damage.
	The genes were named UvrA, UvrB and UvrC for UV resistance.
	Using cell-free extracts, Sancar determined the mechanism of uvrABC which has been refined over the years.


	A disease in humans known as Xeroderma Pigmentosum XP is a rare inherited disease of humans which, among other things, predisposes the patient to
	pigmented lesions on areas of the skin exposed to the sun and
	an elevated incidence of skin cancer.

	It turns out that XP can be caused by mutations in any one of several genes - all of which have roles to play in NER.
	James Cleaver went around and collected cells from hundreds of these patients. He then figured out that the disease was made up of eight genes named XP-A through XP-G plus one called XP-V for variant.


	XP-A participates in photoproduct recognition and DNA binding This binding may be followed by the formation of a quasi-stable complex consisting of XPA, XPC, human single-strand binding protein (RPA/HSSB), and TFIIH, which then acts as a nucleation site for binding of the incision/excision enzymes.
	XP-B is a 3’-> 5’ DNA helicase that may be involved in unwinding the DNA 5'-ward of a damaged base
	XP-C is a single-stranded DNA binding protein that is essential for repair of the nontranscribed regions of the genome, that acts in the initial step of damage recognition.
	XP-D is a 5'–3' helicase, a component of transcription factor TFIIH may be involved in 3'-ward unwinding of the DNA in the vicinity of a damaged base


	XP-E is thought to be involved with the recognition of damaged DNA because it has the capacity to bind to UV-damaged DNA
	XP-F in association with the ERCC1 protein, incises DNA on the 5' side of the damaged site
	XP-G incises DNA 3' to the damaged site
	XP-V protein is a low-fidelity class Y DNA polymerase, that can replicate UV-induced pyrimidine dimers in vivo with the insertion of the correct bases in the daughter strand
	CSA likely participates in a CSB/RNA polII complex stalled at damaged sites in transcriptionally active DNA that helps remove the stalled RNA polII from the DNA damage site.
	CSB is believed to be a DNA helicase that is required for ubiquitinating RNA polII for its remove and degradation at sites of DNA damage.


	XPA, which encodes a protein that binds the damaged site and helps assemble the other proteins needed for NER.
	XPB and XPD, which are part of TFIIH. Some mutations in XPB and XPD also produce signs of premature aging.
	XPF, with ERCC1 cuts the backbone on the 5' side of the damage
	XPG, which cuts the backbone on the 3' side.
	XPC interacts with HR23B in GGR and recognizes damage
	XPD is a DNA helicase in in transcription complex
	VPV is a by-pass polymerase


	Mutagens are any reagent that causes changes in DNA (often referred to as DNA damage) that can ultimately lead to a change in the DNA sequence.
	Examples of endogenous reagents are; free radicals generated during oxidation reactions, pH changes that can lead to changes in DNA, errors in DNA replication and recombination errors.
	Examples or exogenous reagents are UV radiation, ionizing radiation, chemicals such as benzopyrene and and natural compounds such as aflatoxin.


	Mutagens primarily affect DNA by causing a physical change in the structure, which ultimately alters the sequence, leading to changes in genes such that the information is altered. This leads to loss of a protein, a change in the sequence (and likely structure) of a protein or a change in the level of proteins found in cells.


	Many compounds that enter out cells are lipophilic (typically organic compounds). These compounds are not reactive with DNA. A system of enzymes called P450 monooxygenases add oxygen molecules in order to make them more soluble but this also makes them reactive with DNA.


	Xenobiotics are chemical compounds that do not belong to the normal composition of the human body. These compounds enter the body via the diet, air and medication. The principal route of elimination of xenobiotics from the body is biotransformation. They are eliminated by microsomal phase I and microsomal and cytosolic phase II drugmetabolising enzymes. These enzymes add functional groups to make lipophilic molecules more hydrophilic and hence easier to eliminate. The oxidative reactions are mainly catalysed by cytochrome P450 (CYP or P450) enzymes. The CYP superfamily of microsomal hemoproteins catalyses the monooxygenation of a large number of endogenous and exogenous compounds. They play a key role in the
	metabolism of a wide variety of xenobiotics, such as drugs, pesticides and (pre)carcinogens.


	Replication errors
		Polymerase misincorporation
	Recombination errors
		Unequal crossing over, etc.
	Spontaneous base damage
		Deaminations, depurinations
	Byproducts of metabolism
		Oxygen radicals


Chemicals
	Natural
		In foods, e.g. aflatoxin
	Man-made/man-increased
		Nitrogen Mustard - WWI nerve gas
		Benzopyrene - smoke from coal, autos, cigs
Ultraviolet (UV) Radiation (from sun)
Ionizing radiation
	Natural: radon gas, cosmic rays
	Man-made: x-rays, nuclear tests


	Polymerase proofreading
	DNA mismatch repair
	Uracil DNA glycosylase
	Nucleotide excision repair


	Are proteins that replicate DNA
	Have multiple domains or subunits
	A good polymerase domain has a misincorporation rate of 10^-5(1/100,000)
	Any misincorps are clipped off with 99% efficiency by the “proofreading” activity of the polymerase


	Carried out by a multi-protein complex
	Removes bulky adducts from DNA, e.g.
		Pyrimidine dimers caused by UV
		Benzopyrene-DNA adducts
	Nearby nucleotides are also excised
	Resulting single-strand gap is filled in by polymerase







	5’--------CCGAAttCAG-------3’
	3’--------GGCTTAAGTC-------5’


	5’--------CCGAAttCAG-------3’
	3’--------GGCTTAAGTC-------5’
	INCISION
	5’------- CCGAAttCAG ------3’
	3’--------GGCTTAAGTC-------5’
	EXCISION
	5’-------- -------3’
	3’--------GGCTTAAGTC-------5’
	GAP -FILLING (BY A POLYMERASE)
	5’--------CCGAATTCAG-------3’
	3’--------GGCTTAAGTC-------5’


	Xeroderma Pigmentosum
	Autosomal recessive, multigenic, very rare
	Symptoms:
		Dry scaly skin (xeroderma)
		Freckling; pigmentation abnormalities (pigmentosum)
		Extreme sensitivity to sunlight
		Greatly increased incidence of skin cancer (1000 X)
		Neurological abnormalities
	Defect in nucleotide excision repair