| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
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Reading: Brown,
13: 330-353
.....(much of the material on Mutations should be review from
Genetics)
Outline:
Organisms evolve by way of changes in
the Nucleotide Sequence of their Genome.
This Genome Evolution occurs mainly via Mutation
Events, changes in the nucleotide sequence of a given DNA
molecule, and via Recombination Events, the breakage and
rejoining of DNA molecules, either different or the same, to form
new DNA molecules, with usually large changes in the nucleotide
sequence of one or more of the DNA molecules.
See Brown, Fig 13.1
Mutational Events in DNA molecules most often occur as the result of DNA damage, and will be considered here mainly in terms of DNA Repair of such DNA damage.
A. DNA Mutation and Damage: ... [Brown, Section 13.1]
1. Types of Mutations: ... [ Brown, Fig
a. Nucleotide Substitutions: single base changes, with no loss
or gain of base pairs
Also known as Point Mutations
b. Deletions and Insertions: loss or gain of one or more base pairs
c. Gross mutational events: chromosome aberrations, deletions, duplications, transpositions between chromosomes
2. Types of Nucleotide Substitution Mutations
a. Classes of Point Mutations: Transitions vs Transversions ... [Brown, Box 13.1]
Transitions: Pyrimidine <--> Pyrimidine;
Purine <--> Purine
AT --> GC, TA --> CG, ...
Transversions: Purine <---> Pyrimidine
AT --> TA (NOT the same!), AT --> CG, TA --> GC, ...
b. Tautomeric Shifts ...
Spontaneous Point Mutations:
... [Brown, Fig 13.4]
Tautomeric Shifts ... Keto: =O, =NH <--> Enol:
-OH, -NH2
Enol form of Thymine <--- pairs with ---> Guanine
Enol form of Cytosine <--- pairs with ---> Adenine
Tautomeric Shifts with DNA
containing 5-Bromo-Uracil: ... [Brown, Fig 13.6]
5-BU "mimics"
Thymine and can be incorporated into DNA via DNA replication.
However, the rate of Tautomeric
Shifts with 5BU is higher than with Thymine
Hence, 5BU is a Mutagen (causes mutations).
These mutations are usually Transitions and can be either
a GC -> AT or an AT -> GC transition, depending on whether
a mistake was made during Incorporation of the 5BU or during
Replication of the 5BU DNA:
Incorporation Error:
5BU is incorporated opposite a G, and then replicates correctly,
bringing in an A:
GC --> AT transition
Replication Error:
5BU is incorporated
correctly opposite an A, but then replicates incorrectly, bringing
in a G:
AT --> GC transition
c. Oxidative deamination: C -> U ... [ Brown, Fig 13.7]
Oxidative deamination results in Transitions, of either type: AT -> GC or GC -> AT.
3. Single Base Insertions or Deletions:
a. Intercalation Agents,
eg EtBr or Proflavine ... [Brown, Fig 13.8]
Single base insertions
or deletions occur as a result of Slippage during DNA synthesis
... [Brown, Fig 13.5]
4. Mutations resulting from DNA Damage:
DNA Damage results in Structural Distortions, eg Pyrimidine Dimers; A:G mismatch; Alkylation
Pyrimidine Dimer: Adjacent pyrimidines in same strand
form, eg T-T dimer
cyclobutane bridge ... distorts DNA helical structure ... [Brown,
Fig 13.9]
Guanine methylation
Depending on the Damage and the DNA Repair process, mutations that result can be either Transitions or Transversions
5. Damage and Human Genetic
Disease: ... [Journal Article 4; Brown, Box 13.2]
Defects in DNA repair
enzymes in humans are often associated with Genetic Disease:
Xeroderma pigmentosum ... first identifications of specific
proteins with specific human disease
In general, all Human
Genetic Disease is associated with Allelic Variation or
Polymorphisms present in a human population ...
The Disease will be due to presence of one or more specific Alleles
in or near specific Genes ...
Examples of Human Disease
from Increased Numbers of Trinucleotide Repeats:
... [Brown, Table 13.1]
B. Recognition problem:
Given "damage" at a base pair, which base is the damaged
one (should be repaired) and which is the correct one?
C. Solutions:
1) Damage distorts the structure, distortion indicates which to repair, eg T-T dimer
2) Damaged base arises from common distructive process, eg U from deamination ... often have specific enzymes that recognize this specific base, eg U glycosylase ... [Brown, Fig 13.19]
3) Damage occurs on specific DNA strand: Mismatch repair just after replication
D. Repair Systems: ... [Brown, Section 13.1.4]
1. Direct Repair:
a. Photoreactivation:
PR enzyme binds Pyrimidine Dimer; cleaves cyclobutane bridge in presence of near UV light; PR enzyme leaves; original DNA structure restored
b. Methyl Transferases: ... [Brown, Page 348]
Methyl groups often transferred directly from DNA bases to enzyme, eg repair of Methyl-G by the Ada protein ... Ada protein, so methylated, self distructs
2. Excision Repair: damaged region of DNA is excised, DNA synthesis restores the original structure and sequence
Two main types: Nucleotide Excision and Base Excision
a. Nucleotide Excision: "cut and patch"
... [Brown, Fig 13.20; Sancar Journal Article 4]
Basic Steps:
1. Damage recognition
and binding: part of "Excinuclease" does this ...
2. Strong binding to DNA and DNA conformation change: Excinuclease
... ATP needed to provide energy
3. Incision: endonuclease nicks on one or both sides of damage
on damaged DNA strand ...
4. Excision ("cut" step): Helicase removes the oligodeoxynucleotide
created between the two nicks
5. Repair DNA Synthesis ("patch" step): DNA polymerase
fills in the gap
6. Joining: DNA ligase seals the nick.
Example: short "Patch" nucleotide
excision repair in E. coli
"Excinuclease": 3 proteins ... UvrA, UvrB, UvrC ...
gene mutations: UV sensitivity
Steps:
1. Damage recognition and binding: 2x UvrA + UvrB
2. DNA distortion: UvrA as ATPase, leaving UvrB attached to DNA
3. Incision: UvrC now attaches, nicks 5 bases 3' to damage; UvrB
nicks 8 bases 5' to damage
4. Excision: helicase HelII removes 12-13 mer using ATP for energy;
UvrC leaves
5. Repair DNA Synthesis: DNA PolI fills in the 12-mer gap
6. Joining: DNA ligase seals the nick.
Other Nucleotide Excision
in E. coli:
Nick can be inserted on only one side of the damage.
Then an Exonuclease (ExoVII or 5'->3' Exo of PolI) removes
nucleotides including damage
Repair by PolI or PolIII, ligation by DNA ligase.
Human example: ... [Sancar Journal Article 4]
0. "Excinuclease" composed of some 17 proteins ...
many are gene products of genes implicated in human disease, Xeroderma
pigmentosum (XP) in particular but also Cockayne's syndrome (CS)
and trichothiodystrophy (TTD).
1. Damage recognition: XPA protein
1a. Initial Binding to damage: XPA, XPF, RFA (Replication Factor
A)
2. DNA distortion: TFIIH (Transcription Factor II H), XPC ...
requires ATP for energy
TFIIH contains XPB and XPD plus 6 other polypeptides.
3. Incision: XPG joins complex, nicks 5 bases 3' to damage ...
XPF nicks 24 bases 5' to damage
4. Excision ("cut" step): both XPB and XPD as part of
TFIIH have Helicase activity ... 29-mer removed
All of the above proteins constitute
the "Excinuclease" determined to date in humans ...
5. Repair DNA Synthesis: PCNA and RFC from replication, using
ATP for energy, displace the Excinuclease and prepare the gap
for DNA synthesis.
DNA synthesis usually done by Pol-Epsilon, but Pol-Delta can also
do such repair.
6. Joining: DNA ligase seals the nick.
Comparable proteins and mechanisms have been found and demonstrated in Yeast.
b. Transcription-coupled
nucleotide excision repair:
... [Hanawalt Journal Article 4]
During transcription a specific
coupling exists between the transcription RNA polymerase complex
and nucleotide excision enzymes so that:
1) damage in DNA regions encoding expressed genes is preferentially
repaired, and
2) the transcribed strand in such regions is preferentially repaired.
Mechanism: unknown in detail.
Current thinking: Hanawalt Journal Article 4
Bacteria:
1. A Transcription-repair coupling factor has been isolated
that releases RNA polymerase blocked at a lesion ... Thus, transcription
events appear to "start over" in prokaryotes when DNA
damage is encountered.
Humans:
1. HoloRNApolII transcription complex stalls at damage in DNA
2. TFIIH is then already present at the sight of the DNA damage
... this may facilitate "recruitment" of the complete
Nucleotide Excision"Excinuclease" complex ...
3. Best if HoloRNApolII does not abort the transcription event,
since such takes so long for many eukaryotic genes ... may be
able to "back off" via transcription factor SII,
exposing the DNA damage for repair ... following repair, HoloRNApolII
can continue and finish transcription event.
Humans suffering from Cockayne's
syndrome (CS) are defective in Transcription-coupled repair.
CS patients have mutations at one of two loci: CS-A and CS-B.
Products unknown, but one could be TF SII ...
Mice defective in BRCA1, a mouse and human gene mutants in which cause susceptibility to breast and ovarian cancer, appear to be defective in transcription-coupled repair of oxidative DNA damage !!
c. Base Excision: ... [Brown, Fig 13.19]
Steps:
1. Base specific Glycosylase
removes "bad" base, eg Uracil Glycosylase
2. AP nuclease (A-Purinic
or A-Pyrimidinic DNA: base missing) recognizes DNA with missing
base, introduces nick 5' to this damage
3. Exonuclease
removes damaged DNA ("cut")
4. Subsequent repair steps similar or identical
to those of Nucleotide Excision Repair.
Example: E. coli Endo II is both AP endonuclease
and Exonuclease
Polymerase fills in gap ("and patch"), Ligase seals
nick
d. Mismatch Repair ... E.
coli mutHLS system
... [Brown, Figs 13.21, 13.22, 13.23; Modrich Journal Article
4]
Nucleotide Excision with specificity of Strand Selection ...
Strand Selection in Prokaryotes containing a DAM methylase
(DNA-Adenine Methylase):
The adenine residues in GATC sites in DNA are methylated by the
Dam Methylase in a post-replication DNA modification reaction.
Thus, immediately after replication, GATC sites are hemimethylated,
i.e. the adenine in the GATC site in the original, parental DNA
strand are methylated but the adenine in the daughter DNA strand
is not methylated. This difference in presence of a Methyl group
between parental and daughter DNA strands provides the basis for
Strand Selection for Mismatch Repair in these organisms.
Steps in prokaryotes with
a Dam Methylase, e.g. E. coli MutHLS system:
1. Recognition: MutS recognizes and binds the distortion produced by
a Mismatch in a Base Pair
2. MutL binds MutS
3. Strand Selection: The MutL-MutS complex activates an endonuclease
activity associated with MutH
4. MutH recognizes hemimethylated GATC sites on either side of
the Mismatch and introduces a nick in the daughter DNA strand,
i.e. the DNA strand with the non-methylated adenine.
(5. Some evidence suggests that MutH does this by first binding
to MutL-MutS, forming a MutL-MutS-MutH complex, which moves along
the DNA from the Mismatch to the first GATC site)
6. Excision: helicase HelII (UvrD or MutU gene product) unwinds
the DNA from the nick at the GATC site back to the Mismatch. An
appropriate exonuclease, working with HelII, excises the DNA from
the daughter strand.
If the nick was introduced into a GATC site found on the 5' side
of the mismatch when determined along the daughter DNA strand,
then ExoVII or the RecJ exonuclease excises daughter DNA, in a
5'->3' direction (see Figure in Modrich Mismatch Repair paper).
If the nick was introduced into a GATC site found on the 3' side
of the mismatch when determined along the daughter DNA strand,
then ExoI exonuclease excises daughter DNA, in a 3'->5' direction
(see Figure in Modrich Mismatch Repair paper).
7. DNA synthesis ("patch" step): the gap is filled in
by HoloPolIII, in the usual 5'->3' polymerization reaction
which is only the leading strand reaction. HoloPolIII is used
here rather than PolI, because the GATC site can be very far (~
kb) from the site of the mismatch.
8. Ligase seals the nick.
Eukaryotes have a comparable Mismatch Repair system, e.g. humans encode proteins which are homologs of MutS and MutL. Humans do NOT encode a Dam Methylase and Strand Discrimination mechanisms are not understood.
In humans with Hereditary Nonpolyposis Colorectal Cancer (HNPCC), a common colon cancer, the primary step in development of HNPCC tumours is attributable to a defect in one of four loci encoding homologs to MutL or MutS.
Note the Names of these Proteins and their Cognate Genes: Mut
and mut
Cells with mutations in these genes have high spontaneous
mutation rates
True also for other genes involved in DNA metabolism
eg dnaQ - encodes the epsilon subunit of PolIII, the 3'->5'
proofreading Exonuclease
Corollary: nearly all of the
above Repair Systems are Error-Free:
They show the same high fidelity of DNA synthesis as found in
DNA replication
3. Post-Replication Recombination-Mediated Repair
Steps in Post-Rep Rec-Mediated
Repair:
1. DNA replication fork encounters
DNA damage, but slowly moves on by
2. Fork leaves a Gap in
daughter strand opposite damage in Parental Strand, eg an Okazaki fragment is missing
3. Generalized homologous Recombination
mechanisms fill in this Gap with DNA from the Parental Strand
of the Sister Chromatid, or Sister Daughter Chromosome:
A nick is introduced, RecA protein mediates strand assimilation by the damaged chromosome into the gap, second nick is introduced:
Sister Chromatid Exchange
Gaps are now opposite
good templates; these are Repaired as usual: PolI and Ligase
Three Properties:
1) Process is Error Prone ... This type of repair accounts for UV mutagenesis
2) RecA protein required in its role in Strand Assimilation in Homologous Recombination
2) RecA plays a second role, in Induction of the SOS Response
4. SOS Response: ...[Brown, Page 345]
SOS Response: the induction of at least 11 E. coli genes in response to DNA damage
Steps:
1. DNA
damage, probably ssDNA or a DNA degradation product, activates
RecA
2. Activated RecA protein interacts with LexA
repressor and a few other repressors, activating proteolytic activities
of these repressors.
3. The activated proteolytic
activities of these repressors cause them to self distruct
4. Genes controlled by
these repressors are now expressed.
Expression of the genes controlled by LexA repressor is the SOS Response.
Genes expressed in the SOS
Response:
1. the recA and lexA genes
themselves ... hence, RecA-mediated Post Replication repair is
enhanced
2. the uvrA and uvrB genes
... hence, Nucleotide Excision is enhanced
3. the din genes ... Damage
INducible genes
Other inactivated repressors:
1. the umuD repressor:
this in turn induces the umuB and umuC genes, genes whose products
are required for Error Prone repair and mutation events
2. the phage lambda
CI repressor: this induces phages lambda if the E. coli is
a lambda lysogen
"Turn off" of
the SOS Response:
1. DNA damage is repaired
2. RecA protein returns
from "activated" state to "unactivated" state
3. New LexA repressor is
active and represses the SOS-induced genes.
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith