Homologous recombination

it gives a detailed overview of homolgous recombination at molecular level.

1. . . CHAUDHARY DEVI LAL UNIVERSITY, SIRSA SUBMITTED TO: SUBMITTED BY: Miss Gitanjali Ankush Yadav Botany department ROLL NO. : 25

2. HOMOLOGOUS RECOMBINATION

3. INTRODUCTION Recombination is the term generally used to describe outcome of crossing-over between pairs of homologous chromosomes during meiosis. In the 1960s , models were proposed for the molecular events that underlie crossing-over , and it was realized that a key part of molecular recombination is the breakage and subsequent rejoining of DNA molecules . Biologists now use “recombination” to prefer a variety of processes that involves the breakage and union of polynucleotides . These include :

4. Homologous recombination , also called general recombination, which occurs between segments of DNA molecules that share extensive homology. These segment might be present on different chromosomes, or might be two parts of a single chromosome. Homologous chromosome is responsible for crossing-over during meiosis. Site specific recombination , which occurs between DNA molecules that have only a short region of sequence similarity, possibly a few base pairs . Site specific recombination is responsible for the insertion of page genome into bacterial genome . Transposition , which result in transfer of a segment of DNA from one position in the genome to another.

5. Reference: Molecular biology of the gene (seventh addition) 2012

6. MODELS FOR HOMOLOGOUS RECOMBINATION THE HOLIDAY MODEL : Alignment of two homologous DNA molecules. By homologous we mean that the DNA sequences are identical or nearly identical for a reason of at least 100 base pairs or so. Despite this high degree of similarity, DNA molecules can have small region of sequence difference and, may for example, carry different sequence variants known as alleles , of the same gene. Reference: Molecular biology of the gene (seventh addition) 2012

7. 2. Introduction of breaks into the DNA. The break occur in one DNA strand or involve both DNA strands. 3. Formation of initial short regions of base pairing between the two recombinant molecules. This pairing occur when a single stranded region of DNA originating from one parental molecule pairs with its complementary stand in homologous duplex duplex DNA molecule. This step is known as strand invasion . As a result of strand invasion, the two DNA molecules become connected by crossing DNA strands. This structure is called a Holliday Junction . Reference: Molecular biology of the gene (seventh addition) 2012

8. Cleavage of holiday junction. Cutting the DNA strands within the holiday junction regenerates two separate duplex DNA molecules, and therefore finishes genetic exchange. This process is called resolution . If the two DNA molecules are not identical but, for example, carry a few small sequence differences, as is true often between two alleles of the same gene- branch migration through these regions of sequence difference generates DNA duplex carrying one or a few sequence mismatches (see B and b alleles) . Such regions are called heteroduplex DNA . Reference: Molecular biology of the gene (seventh addition) 2012

9. Holiday junction cleavage Reference: Molecular biology of the gene (seventh addition) 2012

10. THE DOUBLE-STRAND BREAK REPAIR MODEL The initiating event is the introduction of a double strand break (DSB) in one of the two DNA molecules. After introduction of the DSB, a DNA-cleaving enzyme sequentially degrades the broken DNA molecule to generate regions of single-stranded DNA ( ssDNA ). This processing creates single-strand extensions, known as ssDNA tails, on the broken DNA molecules; these ssDNA tails terminate with 30 ends. Reference: Molecular biology of the gene (seventh addition) 2012

11. The ssDNA tails generated by this process then invade the unbroken homologous DNA duplex. The invading strand base-pairs with its complementary strand in the other DNA molecule. Because the invading strands end with 3′ termini, they can serve as primers for new DNA synthesis. Elongation from these DNA ends—using the complementary strand in the homologous duplex as a template serves to regenerate the regions of DNA that were destroyed during the processing of the strands at the break site. Reference: Molecular biology of the gene (seventh addition) 2012

12. If the two original DNA duplexes were not identical in sequence near the site of the break (e.g., having single-base-pair changes as described above), sequence information could be lost during recombination by the DSB-repair pathway. The two Holliday junctions found in the recombination intermediates generated by this model move by branch migration and ultimately are resolved to finish recombination

13. HOMOLOGOUS RECOMBINATION PROTEIN MACHINES Organisms from all branches of life encode enzymes that catalyze the biochemical steps of recombination. In some cases, members of homologous protein families provide the same function in all organisms. In contrast, other recombination steps are catalyzed by different classes of proteins in different organisms but with the same general outcome

14. Prokaryotic and eukaryotic factors t hat catalyze recombination steps : Recobination step E. coli protein catalyst Eukaryotic Protein catalyst Pairing homologous DNAs and strand invasion RecA protein Rad51 Dcm 1 (in meiosis ) Introduction of DSB None Spo11 (in meiosis) HO ( for mating type switching) Processing of DNA breaks to generate single strands of invasion RecBCD Helicase /nuclease MRX Protein (also called Rad50/58/60 nucleases ) Assembly of strand exchange protein RecBCD and RecFOR Rad 52 and Rad59 Holiday junction recognition and branch migration RuvAB complex unknown Resolution of holiday junction Ruvc Perhaps Mus81 and others Reference: Molecular biology of the gene (seventh addition) 2012

15. The RecBCD Helicase /Nuclease Processes Broken DNA Molecules for Recombination The RecBCD enzyme processes broken DNA molecules to generate these regions of ssDNA . RecBCD is composed of three subunits (the products of the recB , recC , and recD genes) and has both DNA helicase and nuclease activities. The activities of RecBCD are controlled by specific DNA sequence elements known as Chi sites. The RecB and RecD subunits are both DNA helicases , that is, enzymes that use ATP hydrolysis to melt and unwind DNA base pairs. RecB subunit contains a 3' to 5' helicases and has also a multifunctional Nuclease domain that digests the DNA as it moves along. RecD is a 5' to 3' helicase, and RecC functions to recognize Chi sites. Reference: Molecular biology of the gene (seventh addition) 2012

16. The nuclease activity of RecBCD frequently cleave each strand during unwinding and thereby destroys the DNA. Upon encountering the chi sequences the nuclease activity of the RecBCD enzyme is altered. After encounter with chi site, the other DNA strand (the one with 5' to 3' polarity) is cleaved even more frequently then it was prior to chi site. As a result of this change in activity, the DNA duplex now has a 3' single-strand extension terminating with the Chi sequence at the 3' end. Reference: Molecular biology of the gene (seventh addition) 2012

17. The ssDNA tail generated by RecBCD must be coated by the RecA protein for recombination to occur. To ensure that RecA , rather than SSB, binds these ssDNA tails, RecBCD interacts directly with RecA and promotes its assembly. RecA Protein Assembles on Single-Stranded DNA and Promotes Strand Invasion These proteins catalyze the pairing of homologous DNA molecules. Pairing involves both the search for sequence matches between two molecules and the generation of regions of base pairing between these molecules. The active form of RecA is a protein–DNA filament . The filament grows by the addition of RecA subunits in the 5' to 3' direction, such that a DNA strand that terminates in 3 ends is most likely to be coated by RecA .

18. RecA–ssDNA complex is the active form that participates in the search for a homology. This homology search is promoted by RecA because the filament structure has two distinct DNA-binding sites: a primary site (bound by the first DNA molecule) and a secondary site This secondary DNA binding site can be occupied by double-stranded. A sequence match of just 15 bp provides a sufficient signal to the RecA filament that a match has been found and thereby triggers strand exchange. Once a region of base-pair complementarity is located, RecA promotes the formation of a stable complex between these two DNA molecules. This RecA -bound three-stranded structure is called a joint molecule and usually contains several hundred base pairs of hybrid DNA. Strand exchange thus requires the breaking of one set of base pairs and the formation of a new set of identical base pairs.

19. Model of two steps in the search for homology and DNA strand exchange within the RecA filament Reference: Molecular biology of the gene (seventh addition) 2012

20. The displacement reaction can occur between DNA molecules in several configurations and has three general conditions: One of the DNA molecules must have a single-stranded region. One of the molecules must have a free 3′ end. The single-stranded region and the 3′ end must be located within a region that is complementary between the molecules. RecA promotes the assimilation of invading single strands into duplex DNA as long as one of the reacting strands has a free end. Refrenece : lewins Genes XII 2018

21. We can divide the reaction that RecA catalyzes between single stranded and duplex DNA into three phases: A slow presynaptic phase in which RecA polymerizes on single stranded DNA. A fast pairing reaction between the single-stranded DNA and its complement in the duplex to produce a heteroduplex joint. A slow displacement of one strand from the duplex to produce a long region of heteroduplex DNA. Refrenece : lewins Genes XII 2018

22. When a single-stranded molecule reacts with a duplex DNA, the duplex molecule becomes unwound in the region of the recombinant joint. The initial region of heteroduplex DNA may not even lie in the conventional double-helical form, but could consist of the two strands associated side by side. A region of this type is called a paranemic joint, as compared with the classical intertwined plectonemic relationship of strands in a double helix. Refrenece : lewins Genes XII 2018

23. Holiday junction must be resolved The proteins involved in stabilizing and resolving Holliday junctions have been identified as the products of the ruv genes in E. coli. RuvA recognizes the structure of the Holliday junction. RuvA binds to all four strands of DNA at the crossover point and forms two tetramers that sandwich the DNA. RuvB is a hexameric helicase with an ATPase activity that provides the motor for branch migration. Hexameric rings of RuvB bind around each duplex of DNA upstream of the crossover point. Refrenece : lewins Genes XII 2018

24. RuvAB displaces RecA from DNA during its action. The third gene, ruvC , encodes an endonuclease that specifically recognizes Holliday junctions. It can cleave the junctions in vitro to resolve recombination intermediates. A common tetranucleotide sequence provides a hotspot for RuvC to resolve the Holliday junction. Refrenece : lewins Genes XII 2018

25. Eukaryotic Genes Involved in Homologous Recombination 1 End Processing/ Presynapsis In mitotic cells, DSBs are produced by exogenous sources such as irradiation or chemical treatment and from endogenous sources such as topoisomerases and nicks on the template strand. During replication nicks are converted to DSBs. The ends of these breaks are processed by exonucleolytic degradation to have single-strand tails with 3′–OH ends. The first step in end processing entails binding of the broken end by the MRN or MRX complex, in association with the endonuclease Sae2 ( CtIP in mammalian cells). MRX – Mre11, Rad50, Xrs2 (Yeast) MRN – Mre11, Rad50, Nbs1 (Mammalian) Rad50 is thought to help hold DSB ends together via dimers connected at the tips by a hook structure that becomes active in the presence of zinc ion. After MRN/MRX and CtIP /Sae2 have prepared the DSB ends and removed any attached proteins or adduct that would inhibit end resection, the ends are resected by nucleases that act in concert with DNA helicases that unwind the duplex to expose single-strand DNA ends. After the DSBs have been processed to have 3′–OH single-strand tails, the single-strand DNA is bound first by the single-strand DNAbinding protein RPA to remove any secondary structure. With the aid of mediator proteins that help Rad51 displace RPA and bind the single-strand DNA, Rad51 forms a nucleofilament . Rad51 is required for all homologous recombination processes except single-strand annealing.

26. Rad50 has a coiled coil domain similar to SMC (structural maintenance of chromosomes) proteins. The globular end contains two ATP-binding and hydrolysis regions (a and b) and forms a complex with Mre11 and Nbs1 (N) or Xrs2 (X). The other end of the coil binds a zinc cation and forms a dimer with another MRX/N molecule. The globular end binds to chromatin. The complex binds to double-strand breaks and can bring them together in a reaction involving two ends and one MRN/X complex (top right figure) or through an interaction between two MRX/N dimers (bottom right figure). Structure for Rad50 and model for the MRX/N complex Refrenece : lewins Genes XII 2018

27. The “head” region of Rad50, bound to Mre11, binds DNA, while the extensive coiled coil region of Rad50 ends with a “zinc hook” that mediates interaction with another MRN complex. The precise position of Nbs1 within the complex is unknown, but it interacts directly with Mre11. MRN/X Complex Refrenece : lewins Genes XII 2018

28. 2. Synapsis : Once the Rad51 filament has formed on single-strand DNA in the DBSR and SDSA processes, a search for homology with another DNA molecule begins and, once found, strand invasion to form a D-loop occurs. Strand invasion requires the Rad54 protein and the related Rdh54/Tid1 protein in yeast, and RAD54B in mammalian cells. Although Rad54, Rdh54, and RAD54B are not DNA helicases , the translocase activity causes local opening of double strands, which may serve to stimulate Dloop formation. 3. DNA Heteroduplex Extension and Branch Migration The proteins involved in this step are not as well defined as those required in the early steps of homologous recombination. D-loop formation results in Rad51 filament being formed on doublestranded DNA. Rad54 protein has the ability to remove Rad51 from double-stranded DNA. This step might be important for DNA polymerase extension from the 3′ terminus.

29. 4.Resolution The search for eukaryotic resolvase proteins has been a long process. Mutants of the DNA helicases Sgs1 of yeast and BLM in humans result in higher crossover rates. These helicases have thus been proposed to normally prevent crossover formation by promoting noncrossover Holliday junction resolution. The end structure is suggested to be a hemicatenane , where DNA strands are looped around each other. This structure is then resolved by the action of an associated DNA topoisomerase : Top3 in the case of Sgs1 and hTOPOIIIα in the case of BLM. In vitro, BLM and hTOPOIIIα can dissolve double Holliday junctions into a noncrossover molecule. Double Holliday junction dissolution by the action of a DNA helicase and topoisomerase . The two Holliday junctions are pushed toward each other by branch migration using the DNA helicase activity. The resulting structure is a hemicatenane where single strands from two different DNA helices are wound around each other. This is cut by a DNA topoisomerase , unwinding and releasing the two DNA molecules and forming noncrossover products. While Refrenece : lewins Genes XII 2018

30. Specialized Recombination Involves Specific Sites Specialized recombination involves a reaction between two specific sites. The enzymes that catalyze site-specific recombination are generally called recombinases . Prominent members of the integrase family are the prototypical Int from phage lambda, Cre from phage P1, and the yeast FLP enzyme. The physical condition of the DNA is different in the lysogenic and lytic states: 1 In the lytic lifestyle, lambda DNA exists as an independent, circular molecule in the infected bacterium. 2 In the lysogenic state, the phage DNA is an integral part of the bacterial chromosome (called the prophage ). Transition between these states involves site-specific recombination: To enter the lysogenic condition, free lambda DNA must be inserted into the host DNA. This is called integration. To be released from lysogeny into the lytic cycle, prophage DNA must be released from the chromosome. This is called excision. Integration and excision occur by recombination at specific loci on the bacterial and phage DNAs called attachment ( att ) sites.

31. For describing the integration/excision reactions, the bacterial attachment site ( att ) is called attB , consisting of the sequence components BOB′. The attachment site on the phage, attP , consists of the components POP′. The sequence O is common to attB and attP . It is called the core sequence, and the recombination event occurs within it. The difference in the pairs of sites reacting at integration and excision is reflected by a difference in the proteins that mediate the two reactions: Integration ( attB × attP ) requires the product of the phage gene int , which encodes an integrase enzyme, and a bacterial protein called integration host factor (IHF). Excision ( attL × attR ) requires the product of phage gene xis , in addition to Int and IHF. Thus, Int and IHF are required for both reactions. Xis plays an important role in controlling the direction; it is required for excision, but inhibits integration. Refrenece : lewins Genes XII 2018

32. Site-Specific Recombination Involves Breakage and Reunion The corresponding strands on each duplex are cut at the same position, the free 3′ ends exchange between duplexes, the branch migrates for a distance of 7 bp along the region of homology, and then the structure is resolved by cutting the other pair of corresponding strands. Refrenece : lewins Genes XII 2018

33. Site specific recombination resembles tropoisomerase activity Integrases use a mechanism similar to that of type topoisomerases in which a break is made in one DNA strand at a time. The basic principle of the system is that four molecules of the recombinase are required, one to cut each of the four strands of the two duplexes that are recombining. The reaction involves an attack by a tyrosine on a phosphodiester bond. The 3′ end of the DNA chain is linked through a phosphodiester bond to a tyrosine in the enzyme. This releases a free 5′–OH end. The free hydroxyl group of each strand then attacks the P–Tyr link of the other strand. The successive interactions accomplish a conservative strand exchange, in which there are no deletions or additions of nucleotides at the exchange site, and there is no need for input of energy. The transient 3′–phosphotyrosine link between protein and DNA conserves the energy of the cleaved phosphodiester bond. Refrenece : lewins Genes XII 2018

34. Recombination Pathways Adapted for Experimental Systems The Cre / lox system is derived from bacteriophage P1. The Cre enzyme recognizes and cleaves lox sites. A construct is designed that is flanked by lox sites, with the Cre gene under control of an inducible promoter that can be turned on by temperature, hormones, or in a tissue-specific pattern. Expression of Cre results in production of the Cre protein; the Cre protein then recognizes and cleaves the lox sites and promotes rejoining of the cut lox sites to leave behind a single lox site, with the material between the lox sites having been excised.

35. Using Cre / lox to make cell type–specific gene knockouts in mice. loxP sites are inserted into the chromosome to flank exon 2 of the gene X. The second copy of the X gene has been knocked out. The mouse formed with this construct is called the loxP mouse. Another mouse, called the Cre mouse, has the cre gene inserted into the genome. Refrenece : lewins Genes XII 2018

36. REFRENCES : Molecular biology of the gene (seventh addition) 2012 lewins Genes XII 2018