Recombination and Transposition - PDF

Recombination and transposition at the molecular level  Homologous recombination Recombination: The exchange of genetic information between DNA molecules. Homologous recombination: The exchange between DNA segments that are similar or identical in their DNA sequences (homologous...

Recombination and transposition at the molecular level  Homologous recombination Recombination: The exchange of genetic information between DNA molecules. Homologous recombination: The exchange between DNA segments that are similar or identical in their DNA sequences (homologous DNA molecules). Eukaryotic chromosomes that have similar or identical sequences frequently participate in homologous recombination (crossover) during meiosis I and occasionally during mitosis. When crossing over takes place between sister chromatids, the process is called sister chromatid exchange (SCE). Because sister chromatids are genetically identical, SCE does not produce a new combination of alleles. Genetic recombination: During meiosis, the cross over occurs between non-sister chromatids of homologous chromosomes results in genetic recombination – the shuffling of genetic material to create a new genetic combination that differs from the original. Genetic recombination is an extremely important genetic process because it increases genetic variation. Bacteria are usually haploid. They do not have pairs of homologous chromosomes. Even so, bacteria also can undergo homologous recombination. How can the exchange of DNA segments occur in a haploid organism? First, bacteria may have more than one copy of a chromosome per cell, though the copies are usually identical. These copies can exchange genetic material via homologous recombination. Second, during DNA replication, the replicated regions may also undergo homologous recombination. In bacteria, homologous recombination is particularly important in the repair of DNA segments that have been damaged. The double-strand break–repair model describes many recombination events 1. Double-strand break formation. This pathway starts with the introduction of a DSB in one of two homologous duplex DNA molecules. The other DNA duplex remains intact. 2. Resection. The 5’ ends on each side of the break are degraded by a DNA- cleaving enzyme to produce two 3’ single stranded tails. 3. Strand invasion and regeneration One of the ssDNA tails invade the unbroken homologous DNA duplex initially and the next one follows. The invading strands base-pair with its complementary strand in the other DNA molecule. 4. Regeneration of the destroyed regions 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. As a result of the strand invasion process, regions of new duplex DNA are generated; this DNA, which often contains some mismatched base pairs, is called heteroduplex DNA. 5. Formation of the Holliday junction. After strand invasion, the two DNA molecules become connected by crossing DNA strands to form a structure that is called a Holliday junction. The two Holliday junctions found in this model. This junction can move along the DNA by the repeated melting and formation of base pairs. This process is called branch migration. Resolution of the Holliday junction Resolution can be achieved by cleavage of the Holliday junction. In the first, cutting the DNA strands within the Holliday junction regenerates two separate duplexes. As we shall see, which of the two pairs of DNA strands in the Holliday junction is cut during resolution has a large impact on the extent of DNA exchange that occurs between the two recombining molecules. In the second (alternative) process (in eukaryotes), resolution is achieved by dissolution, a sort of convergence/collapse mechanism, which we describe in more detail below. Resolving the recombination intermediate with two Holliday junctions Resolution of junction x Resolution of junction y Result Site 1 Site 1 No crossover Site 2 Site 2 No crossover Site 1 Site 2 Crossover Site 2 Site 1 Crossover  Dissolution of double Holliday junctions In the second (alternative) process (in eukaryotes), resolution is achieved by dissolution, a sort of convergence/collapse mechanism,  Homologous recombination protein machines Recombination Step E. coli Protein Catalyst Eukaryotic Protein Catalyst Pairing homologous DNAs and RecA protein Rad51 strand invasion Dcm1 (in meiosis) Introduction of DSB None Spo11 (in meiosis) HO (for mating-type switching) Processing DNA breaks to None MRX protein (also called generate single strands for Rad50/58/60 nuclease) invasion Assembly of strand-exchange RecBCD and RecFOR Rad52 and Rad59 Proteins Holliday junction recognition RuvAB complex Not well characterized and branch migration Resolution of Holliday junctions RuvC Rad51c–XRCC3 complex, WRN, and BLM Gene conversion may result from homologous recombination Gene conversion: Conversion of one allele to the allele on the homologous chromosome. How can homologous recombination account for gene conversion? This can occur by two possible ways: DNA mismatch repair and DNA gap repair A heteroduplex formed during branch migration of a Holliday junction contains a DNA strand from each of the two original parental chromosomes. The two parental chromosomes may contain an allelic difference within this region. In other words, this short region may contain DNA sequence differences. If this is the case, the heteroduplex formed after branch migration will contain an area of base mismatch. Gene conversion occurs when recombinant chromosomes are repaired and result in two copies of the same allele.  Gene conversion by DNA mismatch repair The two parental chromosomes had different alleles due to a single basepair difference in their DNA sequences. During recombination, branch migration creates two heteroduplexes with base mismatches. DNA mismatches will be recognized by DNA repair systems and repaired to a double helix that obeys the AT/GC rule. As a result, two possibilities produce no gene conversion, whereas the other two lead to gene conversion. Gene conversion by gap repair synthesis  Site-specific recombination and transposition of DNA DNA replication, repair, and homologous recombination – all occur with high fidelity – ensure that the genomes of an organism are nearly identical from one generation to the next. Conservative site-specific recombination (CSSR or SSR) and Transpositional recombination (transposition): — rearrange DNA sequences and thus lead to a more dynamic genome structure. Site-specific recombination – recombination between two defined sequence elements. Transposition – recombination between specific sequences and nonspecific DNA sites. Homologous recombination vs. site-specific recombination Homologous recombination – occurs between DNA with extensive sequence homology anywhere within the homology. Strand exchange occurs through the formation of Holliday junction. Site-specific recombination – occurs between DNA with no extensive homology (although very short regions may be critical) only at special sites. Strand exchange occurs by precise break/join events and does not involve any DNA loss or DNA resynthesis.  Site-specific recombination SSR occurs at specific DNA sequences (recombination sites) in the target DNA. Recombination sites: The segment of DNA that carries specific short sequence elements where DNA exchange occurs. Recombination sites carry two classes of sequence elements: sequences specifically bound by the recombinases and sequences where DNA cleavage and rejoining occur. Recombination sites are often quite short, 20 bp or so, although they may be much longer and carry additional sequence motifs and protein-binding sites. Example: The integration of the bacteriophage λ genome into the bacterial chromosome. During λ integration, recombination always occurs at exactly the same nucleotide sequence within two recombination sites, one on the phage DNA and the other on the bacterial DNA. Each recombination site is organized as a pair of recombinase recognition sequences, positioned symmetrically. These recognition sequences flank a central short asymmetric sequence, known as the crossover region, where DNA cleavage and rejoining occur. Because the crossover region is asymmetric (not palindromic), a given recombination site always has a defined polarity.  Site-specific recombinases cleave and rejoin DNA There are two families of site-specific recombinases: ▪ The serine recombinases and ▪ The tyrosine recombinases. When site-specific recombinases cleave the DNA, a covalent protein–DNA intermediate is generated. The covalent protein–DNA intermediate conserves the energy of the cleaved phosphodiester bond within the protein–DNA linkage. As a result, the DNA strands can be rejoined by reversal of the cleavage process. No external energy is needed for DNA cleavage and joining by these proteins. Serine recombinases introduce double-strand breaks in DNA and then swap strands to promote recombination. Tyrosine recombinases break and rejoin one pair of DNA strands at a time. Recombination by a serine recombinase Covalent-intermediate mechanism used by the serine and tyrosine recombinases. Recombination by a tyrosine recombinase  SSR can generate three different types of DNA rearrangements Insertion of a segment of DNA Deletion of a DNA segment Inversion of a DNA segment (Eg: bacteriophage λ DNA integration) A, B, C, D, X, and Y denote the genes that lie within the different segments of DNA. Darker red and blue boxes represent the recombinase-recognition sequences Black arrows show the crossover regions. DNA insertion, deletion, or inversion depends on the organization of the recombinase recognition sites on the DNA molecule or molecules that participate in recombination. Recombination between a pair of inverted sites will invert the DNA segment between the two sites. In contrast, recombination between sites organized as direct repeats deletes the DNA segment between the two sites. Finally, insertion specifically occurs when recombination sites on two different molecules are brought together for DNA exchange.  Biological roles of site-specific recombination Many phage insert their DNA into the host chromosome during infection using this recombination mechanism. Site-specific recombination is used to alter gene expression – inversion of a DNA segment can allow two alternative genes to be expressed. Site-specific recombination helps to maintain the structural integrity of circular DNA molecules during cycles of DNA replication, homologous recombination, and cell division. λ Integrase promotes the integration and excision of a viral genome into the host-cell chromosome The infection of host bacterium by a bacteriophage λ results either in establishment of the quiescent lysogenic state or in phage multiplication, a process called lytic growth. Establishment of a lysogen requires the integration of the phage DNA into the host chromosome. When the phage leaves the lysogenic state to replicate and make new phage particles, it must excise its DNA from the host chromosome. To integrate, the λ integrase protein (λInt, a tyrosine recombinase) catalyzes recombination between two specific sites, known as the attB and attP. The attP and attB sites are highly asymmetric. Both sites carry a central core segment (30 bp), which consist of two λInt-binding sites and a crossover region. Whereas attB consists only of this central core region, attP is much longer (240 bp) and carries several additional protein-binding sites. C, C’, B, and B’ – The core λInt-binding sites. attP has additional protein binding sites which are called P arm (sequences on the left) and P’ arm (sequences on the right). P1, P2, and P1’ – The arm λInt-binding sites. H – the integration host factor (IHF)- binding sites X – The Xis binding sites. F – Fis binding site. Gray area – The crossover regions. For clarity, λInt is not shown in the picture. Note: Not all protein-binding sites are filled during either integrative or excisive recombination. When recombination is complete, the circular phage genome is stably integrated into the host chromosome. As a result, two new hybrid sites, attL (left) and attR (right), are generated at the junctions between the phage and the host DNA. Excisive recombination of Bacteriophage λ requires an additional architectural protein, callled Xis (for “excise”). Xis is a phage encoded protein and is only made when the phage is triggered to enter lytic growth. Xis, binds to specific DNA sequences and introduces bends in the DNA, which stimulate the formation of active protein–DNA complex at attR. This complex then interacts with attL and recombination occurs. DNA binding by Xis also inhibits integration (recombination between attP and attB) which ensures that the phage genome will be free, and remain free, from the host chromosome when Xis is present. The Hin recombinase inverts a segment of DNA allowing expression of alternative genes The Salmonella Hin, a serine recombinase inverts a segment of the bacterial chromosome to allow expression of two alternative sets of genes. hixL and hixR – recombination sites, in inverted orientation with respect to one another. hin – Gene encodes Hin fljB – gene encodes H2 flagellin fljA – gene encodes a transcriptional repressor of the gene for the H1 flagellin. ON orientation: H2 flagellin and the H1 repressor are expressed. These cells have exclusively H2- type flagella on their surface. OFF orientation: Neither H2 nor the H1 repressor is synthesized, and the H1-type flagella are present. Flagella are a common target for the host immune system; by switching between alternative forms, at least bacteria in the population can avoid recognition by the immune system. This class of recombination, relatively common in bacteria, known as programmed rearrangements, which often function to “preadapt” a portion of a population to a sudden change in the environment. Recombinases convert multimeric circular DNA molecules into monomers The chromosomes of most bacteria are circular, as are most plasmids in both prokaryotic and eukaryotic cells. Some viral genomes are also circular. An intrinsic problem with circular DNA molecules is that they sometimes form dimers and even higher multimeric forms during the process of homologous recombination. Because of this multimerization problem, many circular DNA molecules carry sequences recognized by site-specific recombinases. Proteins that function at these sequences are called resolvases because they “resolve” dimers (and larger multimers) into monomers. Clearly, it is essential that these proteins specifically catalyze resolution but not the reverse reaction (conversion of monomers to dimers), which would only make the multimerization problem worse! Specific mechanisms are in place to enforce this directional selectivity on the recombination process. The Xer Recombinase catalyzes the monomerization of bacterial chromosomes and of many bacterial plasmids).  Transposition Transposition: specific form of genetic recombination that moves certain genetic elements from one DNA site to another. Transposable elements (TE)or transposons: The mobile genetic elements. When transposable elements move, they often show little sequence selectivity in their choice of insertion sites. As a result, transposons can insert within genes (completely disrupting gene function) or within the regulatory sequences of a gene (may change the expression). Perhaps not surprisingly, therefore, transposable elements are the most common source of new mutations in many organisms. Transposable elements are present in the genomes of all life-forms. Transposon-related sequences can make up huge fractions of the genome of an organism. For example, more than 50% of the human genome is composed of transposon-related sequences and