Molecular Biology of the Gene, 5/E --- Watson et al. (2004)

Molecular Biology of the Gene, 5/E --- Watson et al. (2004)
Part I: Chemistry and Genetics Part II: Maintenance of the Genome Part III: Expression of the Genome Part IV: Regulation Part V: Methods 3/11/05

Part II: Maintenance of the Genome
Dedicated to the structure of DNA and the processes that propagate, maintain and alter it from one cell generation to the next

Ch 6: The structures of DNA and RNA
Ch 7: Chromosomes, chromatins and the nucleosome Ch 8: The replication of DNA Ch 9: The mutability and repair of DNA Ch 10: Homologous recombination at the molecular level Ch 11: Site-specific recombination and transposition of DNA 3/11/05

CHAPTER 7: Chromosomes, chromatin, and the nucleosome

Consider the structure of DNA within the cell, and the biological relevance of the structure.
DNA is associated with proteins in cells, both prokaryotes and eukaryotes, even viruses Each DNA and its associated proteins is called a chromosome

The importance of packing of DNA into chromosomes
Chromosome is a compact form of the DNA that readily fits inside the cell To protect DNA from damage DNA in a chromosome can be transmitted efficiently to both daughter cells during cell division Chromosome confers an overall organization to each molecule of DNA, which facilitates gene expression as well as recombination

Proteins in chromosome (1)
Half of the molecular mass of eukaryotic chromosome is protein In eukaryotic cells a given region of DNA with its associated proteins is called chromatin The majority of the associated proteins are small, basic proteins called histones. Other proteins associated with the chromosome are referred to as non-histone proteins, including numerous DNA binding proteins that regulate the transcription, replication, repair and recombination of DNA.

Proteins in chromosome (2)
Nucleosomes: regular association of DNA with histones to form a structure effectively compacting DNA

What is the cost (challenge) of compaction of DNA into chromosome?
CHAPTER 7: Chromosomes, chromatin, and the nucleosome What is the cost (challenge) of compaction of DNA into chromosome? How the challenge could be resolved? What are the advantage of the challenge?

OUTLINE Chromosome sequence & diversity
CHAPTER 7: Chromosomes, chromatin, and the nucleosome OUTLINE Chromosome sequence & diversity Chromosome duplication & segregation The nucleosome Higher-order chromatin structure Regulation of chromatin structure Nucleosome assembly

Chromosome sequence & diversity
CHAPTER 7: Chromosomes, chromatin, and the nucleosome Chromosome sequence & diversity Chromosomes Shape: circular or linear Number in an organism is characteristic Copy: haploid, diploid, polyploid Genomes 3/15/05

Chromosome sequence & diversity
Difference in the structures of eukaryotic and prokaryotic cells is a key to better understand the molecular processes of genome maintenance and expression, as well as the differences in these processes between eukaryotes and prokaryotes 3/15/05

Figure 7-1* Chromosome sequence & diversity

Genome & the complexity of the organism
Chromosome sequence & diversity Genome & the complexity of the organism Genome size: the length of DNA associated with one haploid complement of chromosomes Gene number: the number of genes included in a genome Gene density: the average number of genes per Mb of genomic DNA See Table 7-2 to find the relationship 3/15/05

Table 7-2

Genes make up only a small proportion of the eukaryotic genome
Chromosome sequence & diversity Genes make up only a small proportion of the eukaryotic genome Increases in gene size: (1) increase in the sequence of regulatory sequence; (2) presence of introns (splicing) Increases in the DNA between genes (intergenic sequences): (1) unique; (2) repeated See Figure 7-2, 3, 4, 5; Table 7-3 3/15/05

Table 7-3

Figure 7-2

Figure 7-4*

Chromosome duplication & segregation (1) Critical DNA elements
CHAPTER 7: Chromosomes, chromatin, and the nucleosome Chromosome duplication & segregation (1) Critical DNA elements Origins of replication Centromeres Telomeres These elements are not involved in gene expression 3/15/05

Origins of replication
Chromosome duplication & segregation Origins of replication Sites at which the DNA replication machinery assembles to initiate replication; required fro replication 30-40 kb apart on each eukaryotic chromosome Only one origin for prokaryotic chromosome

Chromosome duplication & segregation
Centromeres Required for the correct segregation of the chromosomes after replication Direct the formation of kinetochore (an elaborate protein complex) essential for chrom. segregation One chromosome, one centromere The size varies (200 bp- >40 kb) Composed of largely repetitive DNA sequences

Figure 7-6 Centromeres, origin of replication and telomere are required for eukaryotic chrom. maintenance

(2) Eukaryotic chromosome duplication & segregation occur in separate phases of the cell cycle
Cell cycle: a single round of cell division Mitotic cell division: the chrom. Number is maintained during cell division

Figure 7-10 The eukaryotic mitotic cell cycle

Figure 7-11 The events of S phase

Figure 7-11 The events of M phase
Mitotic spindle

(3) Chromosome structure changes as eukaryotic cells divide
Chromosome duplication & segregation (3) Chromosome structure changes as eukaryotic cells divide M phase: condensed state, completely disentangled from each other G1, S, G2 phases: diffused, significantly less compact. The structure of chrom. changes, e.g. DNA replication requires the nearly complete disassembly and reassembly of the proteins associated with each chromosome

Figure 7-13 Changes in chromatin structure
Chromosome condensation REMEMBER: chromosome is a consistently changing structure (dynamics)

(4) The gap phase of the cell cycle allow time to prepare for the next cell cycle stage while also checking that the previous stage is finished correctly. Think of the regulatory mechanisms might be involved to CHECK the cell condition

(5) Different levels of chromosome structure can be observed by microscopy

Figure 7-13 Forms of chromotin structure seen in EM (electron microscopy)

Nucleosome & histone structures
CHAPTER 7: Chromosomes, chromatin, and the nucleosome The nucleosome Nucleosome & histone structures 3/15/05

(1) Nucleosomes are the building blocks of chromosomes
The nucleosome The nucleosome is composed of a core of eight histone proteins and the DNA (core DNA, 147 bp) wrapped around them. The DNA between each nucleosome is called a linker DNA. Each eukaryote has a characteristic average linker DNA length (20-60 bp)

Figure 7-18 DNA packaged into nucleosome
Six-fold DNA compaction

(2) Histones are small, positively charged (basic) proteins
Five abundant histones are H1 (linker histone, 20 kd), H2A, H2B, H3 and H4 (core histones, kd). The core histones share a common structural fold, called histone-fold domain The core histones each have an N- terminal “tail”, the sites of extensive modifications The nucleosome

Figure 7-19 The core histones share a common structural fold
(1) (2)

(3) Many DNA sequence-independent contacts (
(3) Many DNA sequence-independent contacts (?) mediate interaction between between the core histones and DNA The nucleosome Figure 7-25

(4) The histone N-terminal tails stabilize DNA wrapping around the octamer
Figure 7-26 The histone tails emerge from the core of the nucleosome at specific positions, serving as the grooves of a screw to direct the DNA wrapping around the histone core in a left-handed manner.

Higher-order chromatin structure
CHAPTER 7: Chromosomes, chromatin, and the nucleosome Higher-order chromatin structure How does it form? 3/15/05

Higher-order chromatin structure
(1) Histone H1 binds to the linker DNA between nucleosome, inducing tighter DNA wrapping around the nucleosome Figures 7-28, 29

(2) Nuclear arrays can form more complex structures: the 30-nm fiber (“zigzag model”)
Higher-order chromatin structure Figures 7-30 (40-fold compaction)

(3) Further compaction of DNA involves large loops of nucleosomal DNA
Higher-order chromatin structure Additional fold compaction is required, but the mechanism is unclear The nuclear scaffold model is proposed

Figures 7-32 The higher- order structure of chromatin
Figures 7-32 The higher- order structure of chromatin. (a) A transmission electron micrograph, (b) A model

(3) Histone variants alter nucleosome function
Higher-order chromatin structure (3) Histone variants alter nucleosome function Several histone variants are found in enkaryotes This variants can replace one of the 4 standard histones to form alternate nucleosomes

Figures 7-33 Alteration of chromatin by incorporation of histone variants
CENP-A is associated with the nucleosomes containing centromeric DNA

Regulation of chromatin structure
CHAPTER 7: Chromosomes, chromatin, and the nucleosome Regulation of chromatin structure How? 3/15/05

The interaction of DNA with the histone octamer is dynamic
Regulation of chromatin structure The interaction of DNA with the histone octamer is dynamic There are factors acting on the nucleosome to increase or decrease the dynamic nature The dynamic nature of DNA-binding to the histone core is important for access of DNA by other proteins essential genome expression etc.

Figures 7-34 A model for gaining access to core DNA

Nucleosome remodeling complexes facilitate nucleosome movement
Regulation of chromatin structure Nucleosome remodeling complexes facilitate nucleosome movement A large protein complexes facilitate changes in nucleosome location or interaction with the DNA using the energy of ATP hydrolysis.

Figures 7-35 Nucleosome movement catalyzed by nucleosome remodeling complexes

Regulation of chromatin structure
Modification of the N-terminal tails of the histones alters chromatin accessibility, and specific enzymes are responsible for histone modification

Figures 7-38 Modification of the histone N-terminal tails alters the function of chromatin

CHAPTER 7: Chromosomes, chromatin, and the nucleosome
Nucleosome assembly Nucleosomes are assembled immediately after DNA replication, and the assembly requires histone chaperones 3/15/05

Figures 7-41 The inheritance of histones after DNA replication

Welcome Each of You to My Molecular Biology Class

CHAPTER 8: The replication of DNA
Molecular Biology Course CHAPTER 8: The replication of DNA

Teaching Arrangement Watch animation-Understand replication
CHAPTER 8 The replication of DNA Teaching Arrangement Watch animation-Understand replication Go through structural tutorial- Experience the BEAUTY of the nature QA-comprehensive understanding of Chapter 8 Summary and highlight Key points

General Detailed The Chemistry of DNA Synthesis
CHAPTER 8 The replication of DNA The Chemistry of DNA Synthesis The Mechanism of DNA Polymerase The Replication Fork The Specialization of DNA Polymerases DNA Synthesis at the Replication Fork Initiation of DNA Replication Binding and Unwinding Finishing Replication General Detailed

CHAPTER 8 The replication of DNA
The first part describes the basic chemistry of DNA synthesis and the function of the DNA polymerase

The Chemistry of DNA DNA synthesis requires deoxynucleoside triphosphates and a primer:template junction DNA is synthesized by extending the 3’ end of the primer Hydrolysis of pyrophosphate (PPi) is the driving force for DNA synthesis 3/18/05

Figure 8-3 Substrate required for DNA synthesis

The mechanism of DNA Polymerase (Pol)
CHAPTER 8 The replication of DNA The mechanism of DNA Polymerase (Pol) 3/18/05

DNA Pol use a single active site to catalyze DNA synthesis
The mechanism of DNA Pol A single site to catalyze the addition of any of the four dNTPs. Recognition of different dNTP by monitoring the ability of incoming dNTP in forming A-T and G-C base pairs; incorrect base pair dramatically lowers the rate of catalysis (kinetic selectivity). 3/18/05

Figure 8-3

Distinguish between rNTP and dNTP by steric exclusion of rNTPs from the active site.
The mechanism of DNA Pol Figure 8-4 3/18/05

DNA Pol resemble a hand that grips the primer-template junction
The mechanism of DNA Pol Schematic drawing T7 DNA pol Figure 8-5

Thumb Fingers Palm Figure 8-8

DNA Polymerase-palm domain***
Catalytic sites for addition and removal of dNTPs. Binds to two metal ions that alter the chemical environment around the catalytic site. (how?) 3/18/05

DNA Polymerase-finger domain
Binds to the incoming dNTP, encloses the correct paired dNTP to the position for catalysis Bends the template to expose the only nucleotide at the template that ready for forming base pair with the incoming nucleotide Stabilization of the pyrophosphate

DNA Polymerase-thumb domain
Not directly involved in catalysis Interacts with the synthesized DNA to maintain correct position of the primer and the active site, and to maintain a strong association between DNA Pol and its substrate. 3/18/05

DNA Pol are processive enzymes
The mechanism of DNA Pol Processivity is a characteristic of enzymes that operate on polymeric substrates. The processivity of DNA Pol is the average number of nucleotides added each time the enzyme binds a primer:template junction (a few~50,000).

The rate of DNA synthesis is closely related to the polymerase processivity, because the rate- limiting step is the initial binding of polymerase to the primer- template junction.

Figure 8-9

Exonucleases proofread newly synthesized DNA
The mechanism of DNA Pol The occasional flicking of the bases into “wrong” tautomeric form results in incorrect base pair and mis- incorporation of dNTP. (10-5 mistake) The mismatched dNMP is removed by proofreading exonuclease, a part of the DNA polymerase. How does the exonucleases work? Kinetic selectivity

Figure 8-10

The second part describes how the synthesis of DNA occurs in the context of an intact chromosome at replication forks. An array of proteins are required to prepare DNA replication at these sites.

The replication fork The junction between the newly separated template strands and the unreplicated duplex DNA 3/18/05

Both strands of DNA are synthesized together at the replication fork.
Leading strand Okazaki fragment Replication fork Lagging strand Figure 8-11

The initiation of a new strand of DNA require an RNA primer
The replication fork Primase is a specialized RNA polymerase dedicated to making short RNA primers on an ssDNA template. Do not require specific DNA sequence. DNA Pol can extend both RNA and DNA primers annealed to DNA template

RNA primers must be removed to complete DNA replication
The replication fork A joint efforts of RNase H, DNA polymerase & DNA ligase Figure 8-12

Topoisomerase removes supercoils produced by DNA unwinding at the replication fork
Figure 8-15

DNA helicases unwind the double helix in advance of the replication fork
Hexameric protein (see your CD) Figure 8-13

Single-stranded binding proteins (SSBs) stabilize single-stranded DNA
The replication fork Cooperative binding Sequence-independent manner (electrostatic interactions) Figure 8-14

Replication fork enzymes extend the range of DNA polymerase substrate
The replication fork DNA Pol can not accomplish replication without the help of other enzymes DNA helicase, SSB, primase, DNA topoisomerase

The specialization of DNA polymerases
CHAPTER 8 The replication of DNA The specialization of DNA polymerases 3/18/05

DNA Pols are specialized for different roles in the cell
The specialization of DNA pol Each organism has a distinct set of different DNA Pols Different organisms have different DNA Pols DNA Pol III holoenzyme: a protein complex responsible for E. coli genome replication DNA Pol I: removes RNA primers in E. coli

Polymerase switching: the process of replacing DNA Pol/primase with DNA Pol or DNA Pol.
Table 8-2

Sliding clamps dramatically increase DNA polymerase activity
The specialization of DNA pol Encircle the newly synthesized double-stranded DNA and the polymerase associated with the primer:template junction Ensures the rapid rebinding of DNA Pol to the same primer:template junction, and thus increases the processivity of Pol. Eukaryotic sliding DNA clamp is PCNA

Figure 8-17

Figure 8-19 Sliding DNA clamps are found across all organism and share a similar structure

Sliding clamps are opened and placed on DNA by clamp loaders
The specialization of DNA pol Clamp loader is a special class of protein complex catalyzes the opening and placement of sliding clamps on the DNA, such a process occurs anytime a primer-template junction is present. Sliding clamps are only removed from the DNA once all the associated enzymes complete their function.

DNA synthesis at the replication fork
CHAPTER 8 The replication of DNA DNA synthesis at the replication fork 3/18/05

At the replication, the leading strand and lagging strand are synthesized simultaneously. The biological relevance is listed in P To coordinate the replication of both strands, multiple DNA Pols function at the replication fork. DNA Pol III holoenzyme is such an example.

Figure 8-20 The composition of the DNA Pol III holoenzyme

Figure 8-21*** Trombone model

DNA synthesis at the replication fork
Interactions between replication fork proteins form the E. coli replisome Replisome is established by protein-protein interactions DNA helicase & DNA Pol III holoenzyme, which is mediated by the clamp loader and stimulates the activity of the helicase (10- fold) DNA helicase & primase, which is relatively week and strongly stimulates the primase function (1000-fold). This interaction is important for regulation the length of Okazaki fragments.

DNA Pol III holoenzyme, helicase and primase interact with each other to form replisome, a finely tuned factory for DNA synthesis with the activity of each protein is highly coordinated.

The third part focuses on the initiation and termination of DNA replication. Note that DNA replication is tightly controlled in all cells and initiation is the step for regulation.

Initiation of DNA replication
CHAPTER 8 The replication of DNA Initiation of DNA replication 3/18/05

Specific genomic DNA sequences direct the initiation of DNA replication Origins of replication, the sites at which DNA unwinding and initiation of replication occur. 3/18/05

The replicon model of replication initiation
Initiation of DNA replication The replicon model of replication initiation Proposed by Jacob and Brenner in 1963 All the DNA replicated from a particular origin is a replicon Two components, replicator and initiator, control the initiation of replication 3/18/05

Replicator: the entire site of cis-acting DNA sequences sufficient to direct the initiation of DNA replication Initiator protein: specifically recognizes a DNA element in the replicator and activates the initiation of replication Figure 8-23

Replicator sequences include initiator binding sites and easily unwound DNA 3/18/05

Binding and Unwinding: origin selection and activation by the initiator protein 3/18/05

Three different functions of initiator protein: (1) binds to replicator, (2) distorts/unwinds a region of DNA, (3) interacts with and recruits additional replication factors DnaA in E. coli (all 3 functions), origin recognition complex (ORC) in eukaryotes (functions 1 & 3)

Binding and unwinding Protein-protein and protein-DNA interactions direct the initiation process DnaA recruits the DNA helicase DnaB and the helicase loader DnaC DnaB interacts with primase to initiate RNA primer synthesis, see replisome part for more details. 3/18/05

Figure 8-26*

Binding and unwinding Eukaryotic chromosome are replicated exactly once per cell cycle, which is critical for these organims 3/18/05

Pre-replicative complex (pre-RC) formation directs the initiation of replication in eukaryotes
Binding and unwinding Initiation in eukaryotes requires two distinct steps Replicator selection: the process of identifying sequences for replication initiation (G1 phase), which is mediated by the formation of pre-RCs at the replicator region.

Figure 8-29 pre-RC formation

Origin activation: pre-RCs are activated by two protein kinases (Cdk and Ddk) that are active only when the cells enter S phase. Figure 8-30 pre-RC activation & assembly of the replication fork in eukaryotes

Binding and unwinding Pre-RC formation and activation is regulated to allow only a single round of replication during each cell cycle. Only one opportunity for pre-RCs to form, and only one opportunity for pre-RC activation.

Figure 8-31 Effect of Cdk activity on pre-RC formation and activation

Figure 8-32 Cell cycle regulation of Cdk activity and pre-RC formatin

Finishing replication
CHAPTER 8 The replication of DNA Finishing replication 3/18/05

Type II topoisomerases are required to separate daughter DNA molecules
Finishing replication

The End replication problem (Figure 8-34)
Lagging strand synthesis is unable to copy the extreme ends of the linear chromosome Finishing replication The End replication problem (Figure 8-34)

How telomerase works? (Figure 8-36
Telomerase is a novel DNA polymerase that does not require an exogenous template Finishing replication How telomerase works? (Figure 8-36 How the end problem is eventually resolved? (Figure 8-37)

T1:The Chemistry of DNA Synthesis T2: The Mechanism of DNA Polymerase
CHAPTER 8 The replication of DNA T1:The Chemistry of DNA Synthesis T2: The Mechanism of DNA Polymerase T3: The Replication Fork T4: The Specialization of DNA Polymerases T5: DNA Synthesis at the Replication Fork T6: Initiation of DNA Replication T7: Binding and Unwinding T8: Finishing Replication

Completely understand Animations DNA polymerization (Topics 1 & 2)
CHAPTER 8 The replication of DNA Completely understand Animations DNA polymerization (Topics 1 & 2) DNA replication (Topics 3－5) Action of Telomerase (Topic 8) 3/18/05

Welcome Each of You to My Molecular Biology Class

CHAPTER 9: The mutability and repair of DNA
Molecular Biology Course CHAPTER 9: The mutability and repair of DNA

Different changes of DNA -behavior re-address
Chapter 8: Mutation is bad (death and unhealthy), which needs to be repaired Chapter 9: Recombination is good (diversity in a species- beautiful), which is promoted Chapter 10: Transposition is not bad, because it is not repaired. (benefit?)

The consequence of high rates of mutation
Mutation in germ line would destroy the species Mutation in soma would destroy the individual. Maintenance of the correctness of the DNA sequence is definitely crucial for living organisms. Keeping the error rate as low as is so expensive.

The points that I absolutely do not agree with Waston et al.
Be fair and serious to science The points that I absolutely do not agree with Waston et al. Mutation is not good, it could not be responsible for biodiversity. Transposition is different from mutation because (1) producing mechanism is different; (2) no mechanism to correct it; (2) existing in nature in a well- controlled manner (10-5).

Two important sources for mutation (unavoidable)
Inaccuracy in DNA replication (10-7 is not accurate enough) Errors Chemical damage to the genetic material (environment) Lesions Damage

To repair an error or damage
First, Detect the errors Second, Mend/repair the errors or lesions in a way to restore the original DNA sequence.

Questions to be addressed
How is the DNA mended rapidly enough to prevent errors from becoming set in the genetic material as mutation How does the cell distinguish the parental strand from the daughter strand in repairing replication errors

How does the cell restore the proper DNA sequence when the original sequence can no longer be read?
How does the cell deal with lesions that block replication?

Topic 1: Replication errors and their repair
CHAPTER 9 The mutability and repair of DNA Topic 1: Replication errors and their repair 3/22/05

The nature of mutations
Replication errors and replication The nature of mutations Point mutations: Transitions (pyrimidine to pyrimidine, purine to purine) Transversions (pyrimidine to purine, purine to pyrimidine)

Insertions Deletions Gross rearrangement of chromosome. These mutations might be caused by insertion by transposon or by aberrant action of cellular recombination processes.

Rate of spontaneous mutation at any given site on chromosomal ranges from 10-6 to per round of DNA replication, with some sites being “hotspot” . Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, known as DNA microsatellites. These sequences (1) are important in human genetics and disease, (2) hard to be copied accurately and highly polymorphic in the population.

Some replication errors escape proofreading
Replication errors and replication The 3’-5’ exonuclease activity of replisome only improves the fidelity of DNA replication by a factor of 100-fold. The misincorporated nucleotide needs to be detected and replaced, otherwise it will cause mutation.

Figure 9-2 Generation of Mutation

Talking about the story of E. coli repair system
Mismatch repair removes errors that escape proofreading Replication errors and replication Increase the accuracy of DNA synthesis for 2-3 orders of magnitudes. Two challenges: (1)rapidly find the mismatches/mispairs, (2) Accurately correct the mismatch Talking about the story of E. coli repair system

MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone
MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself

MutS is a dimer. One monomer interacts with the mismatch specifically, and the other nonspecifically. DNA is kinked Figure 9-4 Crystal structure of MutS

MutS-mismatch-containing DNA complex recruits MutL, MutL activates MutH, an enzyme causing an incision or nick on one strand near the site of the mismatch. Nicking is followed by the specific helicase (?) (UrvD) and one of three exonucleases (?).

DNA polymerase III

Detail 1: How does the E. coli mismatch repair system know which of the two mismatched nucleotide to replace? The newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis.

Figure 9-5

Detail 2: Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch. Figure 9-6

Eukaryotic cells also repair mismatches and do so using homologs to MutS (MSH) and MutL (MLH). The underlying mechanisms are not the same and not well understood.

CHAPTER 9 The mutability and repair of DNA
Topic 2: DNA dmage 3/22/05

DNA undergoes damage spontaneously from hydrolysis and deamination
DNA damage Resulted from the action of water

Figure 9-7: Mutation due to hydrolytic damage
Deamination CU Hydrolysis creates apurinic deoxyribose Deamination 5-mC  T

The presence of U and apurinic deoxyribose in DNA resulted from hydrolytic reactions is regarded as unnatural, thus is easily be recognized and repaired. Explaining why DNA contains T instead of U (?) Can 5-mC  T lesion be repaired?

DNA is damaged by alkylation, oxidation and radiation
DNA damage Nitrosamines Reactive oxygen species (O2-, H2O2, OH•) Figure 9-8 G modification

Figure 9-9 Thymine dimer. UV induces a cyclobutane ring between adjacent T.

Gamma radiation and X-rays (ionizing radiation) cause double- strand breaks and are particularly hazardous (hard to be repaired).

Mutations are also caused by base analogs and intercalating agents
DNA damage Base analogs: similar enough to the normal bases to be processed by cells and incorporated into DNA during replication. But they base pair differently, leading to mistake during replication. The most mutagenic base anolog is 5-bromouracil.

Intercalating agents are flat molecules containing several polycyclic rings that interact with the normal bases in DNA through hydrogen bonds and base stacking.

Mechanisms to repair a damage
See Table 9-1 for summary Mechanisms to repair a damage Repair of DNA damage Direct reversal of DNA damage by photoreactivation and alkyltransferase Base excision repair Nucleotide excision repair Recombination (DSB) repairs Translesion DNA synthesis

Topic 3: Repair of DNA damage
CHAPTER 9 The mutability and repair of DNA Topic 3: Repair of DNA damage 3/22/05

Two consequence of DNA damage
Repair of DNA damage Some damages, such as thymine dimer, nick or breaks in the DNA backbone, create impediments to replication or transcription Some damages creates altered bases that has no effect on replication but cause mispairing, which in turn can be converted to mutation.

Mechanisms to repair a damage
See Table 9-1 for summary Mechanisms to repair a damage Repair of DNA damage Direct reversal of DNA damage by photoreactivation and alkyltransferase Base excision repair Nucleotide excision repair Recombination (DSB) repairs Translesion DNA synthesis

Direct reversal of DNA damage
Repair of DNA damage Error-free repair

Photoreactivation Figure 9-11
Monomerization of thymine dimers by DNA photolyases in the presence of visible light.

Methyltransferase Figure 9-12
Removes the methyl group from the methylated O6-methylguanine. The methyl group is transferred to the protein itself, inactivating the protein.

AP endonulease & exonulcease Exonulcease/DNA polymerase/ligase
Base Excision repair enzyme remove damaged bases by a base-flipping mechanism Repair of DNA damage Glycosylase Recognizes the damaged base Removes the damaged base AP endonulease & exonulcease 3.Cleaves the abasic sugars Exonulcease/DNA polymerase/ligase 4. Works sequentially to complete the repair event.

Figure 9-14: base-flipping recognition

Figure 9-13: removes the damaged base and repair

Fail-safe systems (最后保险系统)
Figure 9-15: oxoG:A repair. A glycosylase recognizes the mispair and removes A. A fail-safe glycosylase also removes T from T:G mispairs, as if it knows how T is produced?

Nucleotide Excision repair enzymes cleave damaged DNA on either side of the lesion
Repair of DNA damage Recognize distortions to the shape of the DNA double helix Remove a short single-stranded segment that includes the lesion. DNA polymerase/ligase fill in the gap.

Figure 9-16**

Figure Transcription-couple repair: nucleotide excision repair (NER) system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template TFIIH

Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA
Repair of DNA damage Double-strand break (DSB) repair pathway Details are in chapter 10

Figure 10-4. Damage in the DNA template can lead to DSB formation during replication

FIGURE 10-3 DSB repair model for
homologous recombination

Translesion DNA synthesis enables replication to proceed across DNA damage
Error-prone repair*** Occurs when the above repairs are not efficient enough so that a replicating polymerase encounters a lesion Translesion synthesis is also called a fail-safe or last resort mechanism. Repair of DNA damage

Translesion synthesis is catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the damage site. Translesion polymerase is produced by cell in response to the DNA damage Translesion polymerases are expressed as part of the SOS response pathway.

FIGURE 9-19 Crystal structure of a translesion polymerase
FIGURE Crystal structure of a translesion polymerase. A Y-family polymerase found in many organisms.

FIGURE 9-19 Translesion DNA synthesis in E. coli

Molecular Biology of the Gene, 5/E --- Watson et al. (2004)

Similar presentations

Presentation on theme: "Molecular Biology of the Gene, 5/E --- Watson et al. (2004)"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

Molecular Biology of the Gene, 5/E --- Watson et al. (2004)

Similar presentations

Presentation on theme: "Molecular Biology of the Gene, 5/E --- Watson et al. (2004)"— Presentation transcript:

Similar presentations

About project

Feedback