Ch 6: The structures of DNA and RNA Ch 7: Chromosomes, chromatins and the nucleosome

1. 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

2. Different changes of DNA sequences Listen to the nature • Mutation is bad and naturally repaired, thus it could not be responsible for biodiversity. • Recombination is good and naturally promoted; it is responsible for diversity inside of species. • Transposition 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.

3. CHAPTER 10 Homologous recombination at the molecular level • Molecular Biology Course

4. All DNA is recombinant DNA. • Genetic exchange works constantly to blend and rearrange chromosomes, most obviously during meiosis, when homologous chromosomes pair prior to the first nuclear division. • During this pairing, genetic exchange between the chromosomes occurs. This exchange, classically termed crossing over, is one of the results of homologous recombination.

5. OUTLINE • Models for Homologous Recombination (two models)** • Homologous Recombination Protein Machines (RecBCD pathway)** • Homologous Recombination in Eukaryotes (meiosis recombination, proteins) • Mating-Type Switching (a gene conversion event) • Genetic Consequences of the Mechanism of Homologous Recombination (exchange or conversion)

6. CHAPTER 10 Homologous recombination at the molecular level Topic 1: Models for Homologous Recombination • Holliday model (Holliday模型) • Double-strand break (DSB) repair model (双链断裂修复模型)

7. Key steps shared by different models • Alignment (结盟) of two homologous DNA molecules. • Introduction of breaks in the DNA • Formation of initial short regions of base pairing between the two recombining DNA molecules: strand invasion • Movement of the Holliday junction: branch migration • Cleavage of the Holliday junction: resolution

8. The Holliday model illustrates key steps in homologous recombination The Holliday model very well illustrates the DNA strand invasion, branch migration, and Holliday junction resolution processes central to homologous recombination. Watch the animation for details!

9. The double-strand break repair model more accurately describes many recombination events Homologous recombination is often initiated by double-stranded breaks (DSB) in DNA, while Holliday model is initiate by the pair of aligned nicks. DSB occurs relatively frequently, but the aligned nicks not.

10. DSB breaks Processing of the DSB to generate DNA with 3’ ss tails Strand invasion of 3’ end Second strand invasion and DNA repair synthesis from the 3’ ends Branch formation and migration Figure 10-3 DSB repair model

11. Double –stranded DNA breaks arise by numerous means and initiate homologous recombination DSBs in DNA arise quite frequently. In bacteria, the major biological role of homologous recombination is to repair DSBs (Ionizing radiation, damage in the DNA template); DSBs also promotes genetic exchange. In eukaryotic cells, homologous recombination is critical for DNA repairing, as well as for the process of chromosomal pairing during meiosis.

12. (复制叉瓦解) Figure 10-4. Damage in the DNA template can lead to DSB formation during replication

13. CHAPTER 10 Homologous recombination at the molecular level Topic 2: Homologous recombination protein machines---RecBCD pathway • RecBCD helicase/nuclease processes broken DNA ends for recombination (  site). • RecA protein promotes strand invasion (how?). • RuvAB complex promotes branch migration. • RuvC resolves the Holliday junction

14. Watch the animation for details!

15. CHAPTER 10 Homologous recombination at the molecular level Topic 3: Homologous recombination in eukaryotes

16. Biological functions of homologous recombination in eukaryotes • Critical for DNA repair and the restarting of collapsed (瓦解的) replication forks. • During meiosis, homologous recombination is Required for proper chromosome pairing, thus for maintaining the integrity of the genome.

17. The duplicated homologous chromosomes must pair. *** Without recombination, chromosome often fails to align properly for the first meiotic division. Figure 10-14 DNA dynamics during meiosis

18. Programmed generation of double –stranded DNA breaks occurs during meiosis • The Spo11 protein introduces DSBs in chromosomal DNA at many locations to initiate meiotic recombination. • SPO11 gene is expressed specifically during meiosis, and Spo11-mediated DNA cleavage occurs right around the time when the replicated homologous chromosomes start to pair.

19. Figure 10-16 Mechanism of cleavage by Spo11.

20. MRX protein processes the cleaved DNA ends for assembly of the RecA-like strand –exchange proteins Dmc1 is a RecA-like protein that specifically functions in meiotic recombination. Rad51 has a similar function.

21. Figure 10-17 Meiotic recombination pathway

22. CHAPTER 10 Homologous recombination at the molecular level Topic 4: mating-type switching • Mating-type switching is initiated by a site-specific double-strand break • Mating-type switching is a gene conversion event, not associated with crossing over

23. Figure 10-20 Genetic loci encoding mating-type information of yeast S. cerevisiae (啤酒酵母) HML and HMR loci are called silent cassettes because they are not expressed, which provide a “store house” of genetic information used to switch a cell’s mating type

24. Intramolocular alignments Rad51-dependent strand invasion Synthesis of two new DNA strands from a information from HMRa Branch migration, new DNA synthesis, and branch resolution

25. CHAPTER 10 Homologous recombination at the molecular level Topic 4: Genetic Consequences of the Mechanism of Homologous Recombination (HR) • HR is generally independent of sequence and therefore the frequency of recombination between any two genes is generally proportional to the distance between those genes. ---The molecular basis of Morgen’s chromosome mapping

26. Gene conversionoccurs during HR • Genes close to the sites of DSB is converted during recombination.

27. Gene conversionoccurs during HR 2. Repair of the mismatches in the recombination intermediates.

28. CHAPTER 10 Homologous recombination at the molecular level 重点和难点 Topics 1 & 2 are the keys to understand homologous recombination and other topics.