Mutation and DNA Repair. Mutation Rates Vary Depending on Functional Constraints.

Mutation and DNA Repair

Mutation Rates Vary Depending on Functional Constraints

Low Mutation Rates are Necessary for the Evolution of Complexity

1. Because most mutations are deleterious, there are limits to the number of mutations that an organism can afford to accumulate in its somatic body, e.g.,

a) given mean eukaryotic rates, genomes can accommodate 60,000 genes without intolerable mutational loads (Alberts et al.)

b) a mutation rate 10 times higher would limit genome size to ca. 6000 genes

2. Both the germ line and the somatic body must be protected from mutational load (rare mutations become common because of large genomes and cell proliferation), e.g.,

a) germ line: (1) DNA repair(2) meiotic recombination in all eukaryotes(3) sequestering of germ line in metazoans(4) diplontic selection among cell lineages in meristems of plants

b) somatic tissues ...20% of deaths in western societies are due to cancer (uncontrolled cell proliferation) resulting largely to the accumulation of genetic damage in somatic tissues

(1) DNA repair(2) immune systems

3. less efficient DNA repair and absence of meiosis may explain the limitation of prokaryotes to small genomes and unicellular forms before the origin of these processes in the protoeukaruote line.

4. spontaneous nucleotide changes are much higher than mutation rates would indicate, because of DNA repair mechanisms

Two strategies to study gene function

• Genotype to Phenotype - sequencing and searching for homologous sequences, then study their function

• Phenotype to Genotype - mutational screens and functional analysis

Kinds of Mutations

• Point Mutations– Same sense mutations– Missense Mutations– Nonsense Mutations

– Transitions– Transversions

• Frame shift mutations

• Substitutions, Deletions and Additions

Chemistry of single nucleotide substitutions:

a) transitions: a pyrimidine replaces a pyrimidine (C T or T C)

or a purine replaces a purine (A G or G A)

b) transversions: a pyrimidine replaces a purine or vice versa

c) transitions are less severe mutations that transversions:

(1) chemically, purines are more similar to one another than

they are to pyrmidines, and vice versa

(2) genetically, amino acid substitution is less likely with

transitions because of the degeneracy of the genetic code

(a) 3rd position transitions often code same amino acid

i) UUU and UUG both code for leucine

ii) GAA and GAG both code for glutamic acid

(b) 3rd position transversion less often codes for same

amino acid

i) UUU and UUG code for phenylanaline and leucine

Mutagenesis

• Spontaneous Mutations– Replication Errors– Other Errors

• Chemical Mutagenesis

• Radiation-induced Mutations

Replication Errors

Replication Proofreading

Mutator Strains of E. coli• error prone replication

• mutD codes for subunit of DNA pol III:

DNA polymerase III holoenzyme with subunits

(weight in daltons)

Step 1: previous nucleotide pair is tested for complementarity. If passed, elongation occurs.

Step 2: If failed, the elongating strand is transferred to the exonuclease site to excise the mismatched nucleotide.

Experimental Demonstration of Proofreading

artificial template

double labeled probe

last nucleotide is non-complementary and labeled

non-complementary nucleotide excised, but no complementary

nucleotides

Tautomerization of Bases

Thymine Tautomers: T•A to T•G bindingmutation from T to A

Replication

replication

A•Tketo

A•Tketo

template

daughter

A•Tketo

daughter

template

replication AT

replication AT

replication AT

replication AT

replication

A•Tenol

A•Tketo

template

daughter

G•Tenol

daughter

template

replication AT

replication AT

replication GC

replication AT

if unrepaired

Adenine Tautomers : A•T to A•C bindingmutation from C to T

Replication

replication

Aamino

•T

Aamino

•T

template

daughter

Aamino

•T

daughter

template

replication AT

replication AT

replication AT

replication AT

replication

Aimino

•T

Aimino

•C

template

daughter

Aamino •T

daughter

template

replication AT

replication GC

replication AT

replication AT

if unrepaired

Cytosine Tautomers : Camino•G Cimino•A binding

mutation from C to T

commonresults in

C•G pairing

rareresults in

C•A pairingAT substitution

Replication

replication

Camino

•G

Camino

•G

template

daughter

Camino

•G

daughter

template

replication CG

replication CG

replication CG

replication CG

replication

Cimino

•G

Cimino

•A

template

daughter

Camino •G

daughter

template

replication CG

replication TA

replication CG

replication CG

if unrepaired

Guanine Tautomers : Gketo•C Genol•T binding

mutation from G to A

commonresults in

G•C pairing

rareresults in

G•T pairing

Replication

replication

Gketo

•C

Gketo

•C

template

daughter

Gketo

•C

daughter

template

replication GC

replication GC

replication GC

replication GC

replication

Genol

•C

Genol

•T

template

daughter

Gketo

•C

daughter

template

replication GC

replication TA

replication GC

replication GC

if unrepaired

Frameshift Mutations

insertion

Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication

Spontaneous Mechanisms Outside of Replication

Spontaneous hydrolysis can result in deamination and depurination

Deaminationreplacement of an amino group by a carbonyl oxygen

These nucleotide analogs have different pairing affinities, but analogs can be

recognized and repaired

5-methyl C deamination results in T, which can’t be recognized as a mutation

Replication produces a GC and an AT

C’s are selected for methylation in certain CG sequences, which has led to the conversion of most CG’s to TG’s during evolution

Deamination of C and Aillustrating different pairing behavior

Deamination and repair of C

Deamination

Repair of a Deaminated

Cytosine

Deamination of 5-methylcytosine

Triplet Repeats

Pathology results when repeats exceed a threshold number.

Amplification of copy number by unequal crossing-over

Unequal crossing-over becomes more likely with increased copy number

Dynamic Mutations

Unequal crossing-over becomes more likely with increased copy number

and

The severity of the pathology increases with copy number

therefore...

Both the probability of the pathology and its severity increase over generations after the number of repeats approaches the threshold

A number of conditions are based on this mechanism operating in different genes

The repeats can be located in different orientations with regard to the coding sequence

upstream

downstream

within

within

The repeats can be located in different orientations with regard to the coding sequence ...even within a single gene

Chemical Mutagenesis

EMS is an alkylating agent

Nucleoside analogs can exhibit variant pairing behavior

keto (above); enol pairs to G instead of A

Acridine dyes intercalate DNA sequences

Effect: stabilizes the looping that leads to deletions and insertions that cause

frame shift mutations

Mechanism of Frameshift Mutation: “Slipping a cog” …a base fails to pair with its partner during replication

Major Repair Mechanisms

• Mismatch repair

• Excision repair

• Double strand breaks repaired mainly by end-joining

• Inducible & error-prone mechanisms

Excision Repair

Excision repair mechanism

More excision repair modalities

Repair of UV damage

Thymine dimers

Excision Repair of UV Induced Thymine Dimers

Mismatch Repair

Mismatch Repair

• To catch single-base errors that slip through proofreading during replication

• Happens right after replication

• Misses C•C and small insertions and deletions

• mutH, mutL, mutS mutator strains are involved in mismatch repair

• Trick is distinguishing the new daughter strand

MissmatchRepair

MutH

How is the daughter strand recognized as the strand to correct?

MismatchRepair

GATC sequences methylated on the 6 position of the A base

MismatchRepair endonuclease

Activity

…nicks DNA

and then methylation

Radiation Induced Mutagenesis

• UV induced Thymine dimers

• Gamma and X-ray double stranded breaks

Spectrum

X-rays induce mutations

Multiple mechanisms to repair UV damage

Photo-activatedRepair System

problem: deletion of short nucleotide sequence

Repairing Double-stranded Breaks• often caused by radiation (high energy gamma or X-rays, directly or by creation of free radicals)

• repaired by: ◊ Homologous recombination ◊ Blunt-end repair (right)

Inducible Repair

• Backup systems activated only in emergencies

• Inducible

• Error prone

SOS

Undoing alkylation

Note that the enzyme is expended!

A tangible example of the importance of DNA repair

Photo-activatedRepair System

RecombinationRepair

bulky mutations can leave gapsafter replication

p53 activation of DNA repair