Welcome to My Molecular Biology Class

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 3/22/05

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 3/22/05

The consequence of high rates of mutation Mutation in germ line (生殖细胞) would destroy the species Mutation in soma (体细胞) 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 10-10 (page191) is very expensive.

Build up a serious attitude to science!!! I absolutely do not agree with Waston et al. at the points described 3rd & 4th paragraphs on page 235 What are the more reasonable explanations for the 10-10 mutation frequency in living organisms? What are the evidences that such a low mutation rate can drive the evolvement of a new species if the cell changes are known harmful?

Listening to nature Mutation (突变) is naturally avoided by repair of the errors and damages, suggesting it is generally not welcome in living organisms. Recombination (重组) is good and naturally promoted; it is responsible for diversity inside of species. Transposition (转座) is different from mutation and recombination because (1) producing mechanism is different; (2) no mechanism to correct it; (3) existing in nature in a well-controlled manner (10-5). Not repaired but controlled.

CHAPTER 9: The mutability and repair of DNA Molecular Biology Course 3 teaching hours CHAPTER 9: The mutability and repair of DNA Replication errors and their repair DNA damage Repair of DNA damage

Two important sources for mutation (unavoidable) Inaccuracy in DNA replication (10-7 is not accurate enough) Errors (错误) Chemical damage to the genetic material (environment) Lesions (损害,伤害arose from spontaneous damage) Damage (损害,伤害 caused by chemical agents and radiation

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 How the replication errors are resulted? The nature of the replication errors is mismatch. 2. How mismatches are recognized and correctly repair?

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 10-11 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 (微卫星DNA). These sequences (1) are important in human genetics and disease, (2) hard to be copied accurately and highly polymorphic in the population.

How the replication errors are resulted?

Each bases has its preferred tautomeric form (Ch 6)

The strictness of the rules for “Waston-Crick” pairing derives from the complementarity both of shape and of hydrogen bonding properties between adenine and thymine and between guanine and cytosine.

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 (Fig. 9-2).

Figure 9-2 Generation of Mutation

2. How the replication errors are repaired?

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 (why?) (UrvD) and one of three exonucleases (why?).

Exonuclease, Helicase 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 (自发的) from hydrolysis (水解) and deamination (转氨) 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

Can 5-mC  T lesion be repaired? Vertebrate DNA frequently contains 5-methyl cytosine in place of cytosine as a result of the action of methyl transferase. This modified base plays a role in the transcriptional silencing (Ch 17). The presence of U and apurinic deoxyribose in DNA resulted from hydrolytic reactions is regarded as unnatural, thus is easily be recognized and repaired. Can 5-mC  T lesion be repaired?

DNA is damaged by alkylation (烷基化), oxidation (氧化) and radiation (辐射) DNA damage Alkylating chemical: Nitrosamines (亚硝胺) Reactive oxygen species (O2-, H2O2, OH•) Figure 9-8 G modification

“O2-” hyperoxide “H2O2” Peroxide “OH•” hydroxyl

Figure 9-9 Thymine dimer. UV induces a cyclobutane (环丁烷) ring between adjacent T. Radiation damage 1

Gamma radiation and X-rays (ionizing radiation) cause double-strand breaks and are particularly hazardous (hard to be repaired). Radiation damage 2

Mutations are also caused by base analogs (碱基类似物) and intercalating agents (嵌入剂) 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 mispairing during replication. The most mutagenic base analog is 5-bromoUracil (5-BrU) (溴尿嘧啶).

酮异构体 烯醇异构体 Figure 9-10a Base analogues Figure 3-33 G-U pair

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

溴乙非啶 二氨基吖啶/原黄素 吖啶, 氮蒽 Figure 9-10b Intercalating agents

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 (光活化作用) and alkyltransferase (烷基转移酶) Base excision repair (切割修复) 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 sugar-phosphate backbone Exonulcease/DNA polymerase/ligase 4. Works sequentially to complete the repair event.

Figure 9-14: base-flipping recognition by glycosylase

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 9-17. 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 TFIIH is a transcription factor including XPA and XPD (UvrB)

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

FIGURE 9-19 Translesion DNA synthesis in E. coli

CHAPTER 9 The mutability and repair of DNA Summary and key points All the repair mechanisms (details) and the cause of the corresponding DNA errors and DNA damages Mismatch repair system: DNA replication errors Direct reversal of DNA damages: utraviolet (UV) irradiation induced thymine/pyrimidine dimmers---photoactivation; alkylation agents caused O6-methylguanine---methyl transferase. Base excision repair: the base damage by alkylation and oxidation Nucleotide excision repair: the distortion of the DNA double helix by thymine dimmer or the bulky chemical adduct (加合物) on a base. Recombination repair: double-strand breaks in DNA, errors encountered by a replication fork. Translesion synthesis allows the replication to proceed across DNA damage at a cost of error-prone replication. A different DNA polymerase is utilized.

CHAPTER 9 The mutability and repair of DNA Homework Review the lecture