GENETIC RECOMBINATION AND MAPPING

GENETIC RECOMBINATION AND MAPPING

INTRODUCTION

In the two preceding sections we discussed the mechanisms by which DNA sequences in cells are maintained from generation to generation with very little change. Although such genetic stability is crucial for the survival of individuals, in the longer term the survival of organisms may depend on genetic variation, through which they can adapt to a changing environment. Thus an important property of the DNA in cells is its ability to undergo rearrangements that can vary the particular combination of genes present in any individual genome, as well as the timing and the level of expression of these genes. These DNA rearrangements are caused by genetic recombination. Two broad classes of genetic recombination are commonly recognized : –

(a) General recombination and (b) Site-specific recombination.

In general recombination, genetic exchange takes place between any pair of homologous DNA sequences, usually located on two copies of the same chromosome. One of the most important examples is the exchange of sections of homologous chromosomes (homologues) in the course of meiosis. This "crossing-over" occurs between tightly apposed chromosomes early in the development of eggs and sperm and it allows different versions (alleles) of the same gene to be tested in new combinations with other genes, increasing the chance that at least some members of a mating population will survive in a changing environment. Although meiosis occurs only in eukaryotes, the advantage of this type of gene mixing is so great that mating and the reassortment of genes by general recombination are also widespread in bacteria.

This process leads to offspring having different combinations of genes from their parents and can produce new chimeric alleles. Enzymes called recombinases catalyze natural recombination reactions. RecA, the recombinase found in E. coli, is responsible for the repair of DNA double strand breaks (DSBs). In yeast and other eukaryotic organisms there are two recombinases required for repairing DSBs. The RAD51 protein is required for mitotic and meiotic recombination and the DMC1 protein is specific to meiotic recombination.

Chromosomal crossover refers to recombination between the paired chromosomes inherited from each of one's parents, generally occurring during meiosis. During prophase-I the four available chromatids are in tight formation with one another. While in this formation, homologous sites on two chromatids can mesh with one another, and may exchange genetic information. Because recombination can occur with small probability at any location along chromosome, the frequency of recombination between two locations depends on their distance. Therefore, for genes sufficiently distant on the same chromosome the amount of crossover is high enough to destroy the correlation between alleles. In gene conversion, a section of genetic material is copied from one chromosome to another, but leaves the donating chromosome unchanged.

Recombination can occur between DNA sequences that contain no sequence homology. This is referred to as Nonhomologous recombination or Nonhomologous end joining. DNA homology is not required in site-specific recombination. Instead, exchange occurs at short, specific nucleotide sequences (on either one or both of the two participating DNA molecules) that are recognized by a variety of site-specific recombination enzymes. Site-specific recombination therefore alters the relative positions of nucleotide sequences in genomes. In some cases these changes are scheduled and organized, as when an integrated bacterial virus is induced to leave a chromosome of a bacterium under stress; in others they are haphazard, as when the DNA sequence of a transposable element is inserted at a randomly selected site in a chromosome.

As for DNA replication, most of what we know about the biochemistry of genetic recombination has come from studies of prokaryotic organisms, especially of E. coli and its viruses.

 RECOMBINATION

Independent Assortment and Crossing Over

Independent Assortment - The Law of Independent Assortment, also known as "Inheritance Law", states that the inheritance pattern of one trait will not affect the inheritance pattern of another. While Mendel's experiments with mixing one trait always resulted in a 3:1 ratio between dominant and recessive phenotypes, his experiments with mixing two traits (dihybrid cross) showed 9:3:3:1 ratios (Figure 3.1). 

But the 9:3:3:1 table shows that each of the two genes is independently inherited with a 3:1 ratio. Mendel concluded that different traits are inherited independently of each other, so that there is no relation, for example, between a cat's color and tail length. This is actually only true for genes that are not linked to each other. Independent assortment occurs during meiosis I in eukaryotic organisms, specifically anaphase I of meiosis, to produce a gamete with a mixture of the organism's maternal and paternal chromosomes. Along with chromosomal crossover, this process aids in increasing genetic diversity by producing novel genetic combinations. Of the 46 chromosomes in a normal diploid human cell, half are maternally-derived (from the mother's egg) and half are paternally-derived (from the father's sperm).

This occurs as sexual reproduction involves the fusion of two haploid gametes (the egg and sperm) to produce a new organism having the full complement of chromosomes. During gametogenesis - the production of new gametes by an adult - the normal complement of 46 chromosomes needs to be halved to 23 to ensure that the resulting haploid gamete can join with another gamete to produce a diploid organism. An error in the number of chromosomes, such as those caused by a diploid gamete joining with a haploid gamete, is termed aneuploidy.

In independent assortment the chromosomes that end up in a newly-formed gamete are randomly sorted from all possible combinations of maternal and paternal chromosomes. Because gametes end up with a random mix instead of a pre-defined "set" from either parent, gametes are therefore considered assorted independently. As such, the gamete can end up with any combination of paternal or maternal chromosomes. Any of the possible combinations of gametes formed from maternal and paternal chromosomes will occur with equal frequency. For human gametes, with 23 pairs of chromosomes, the number of possibilities is 2²³ or 8,388,608 possible combinations. The gametes will normally end up with 23 chromosomes, but the origin of any particular one will be randomly selected from paternal or maternal chromosomes. This contributes to the genetic variability of progeny.

Crossing Over - Crossing over occurs between equivalent portions of two nonsister chromatids (Figure 3.2). Each chromatid contains a single molecule of DNA. So the problem of crossing over is really a problem of swapping portions of adjacent DNA molecules. It must be done with great precision so that neither chromatid gains or loses any genes. In fact, crossing over has to be sufficiently precise that not a single nucleotide is lost or added at the crossover point if it occurs within a gene. Otherwise a frameshift would result and the resulting gene would produce a defective product or, more likely, no product at all.

How do nonsister chromatids ensure that crossing over between them will occur without the loss or gain of a single nucleotide? One plausible mechanism for which there is considerable laboratory evidence postulates the following events.

Note that each recombinant DNA molecule includes a region where nucleotides from one of the original molecules are paired with nucleotides from the other. But no matter, the need for a smooth double helix guarantees that each exchange takes places without any gain or loss of nucleotides. So long as the total number of nucleotides in each strand and the complementarities (A-T, C-G) are preserved, this "heteroduplex" region (which may extend for hundreds of base pairs) will only rarely have genetic consequences. And these may, in fact, be helpful because the synthesis of a short stretch of DNA using the template provided by the other chromatid also provides a mechanism for repairing any damage that might have been present on the "invading" strand of DNA. If the cut in the molecule 1 occurs in the region of a mutation, the damaged or incorrect nucleotides can be digested away. Refilling the resulting gap, using the undamaged molecule 2 as the template, repairs the damage to molecule 1. Why should the cutting and ligation be limited to the strands shown? They are not. Half the time the cutting and ligating rejoins the original parental arms. In these cases, no crossover takes place. The only genetic change that might have occurred is a transfer of some genetic information in the heteroduplex region. So crossing over not only provides a mechanism for genetic recombination during meiosis but also provides a means of repairing damage to the genome.

Molecular mechanisms of Recombination

DNA replication with 100% fidelity is a nice feature to keep offspring in just the genetic background of the species. But to get there, or to evolve further, requires genetical changes, one of which results from recombination of (near) homologous parts of DNA. The nature of structural changes in DNA neccessary to result in homologous genetic recombination were layed out by R. HOLLIDAY in 1964, and in subsequent years the crossover-structures were visualized by electron microscopy (Figure 3.3). The actual conformation of a DNA crossover was speculated to be a four-way-junction with separate DNA helices, or with stacked helices in either a parallel or an antiparallel orientation of the helices. The models had to allow for branch migration, else no exchange of genetic material would happen.

Breakage - Fusion (Reunion) - Bridge Cycle, Control Elements And Unstable Genes - Since the beginning of the century has it been known that unstable or variable gene loci occur in plants although the drastically enhanced mutability and the increased back mutation rate could not be explained at first. The decisive breakthrough was accomplished by B. McCLINTOCK with her studies on maize chromosomes published in 1947 and 1951. The basis of these were her earlier observations and analyses (1938) on breakage-fusion (reunion)-bridges. Their occurrence could be correlated with the restructuring in chromosomes.

Bridges are formed during anaphase whenever two chromosomes fuse at their ends generating a fusion product with two centromers. If these two are subsequently torn to different poles then will inevitably occur a chromosomal fraction. During the following S-phase of the interphase nucleus is a chromatid with a fraction at its terminus replicated in just the same way as the other chromosomes leading again to a fusion of the homologous chromatids. Consequently can a chromosome consisting of just one chromatid but two centromers be found in the subsequent mitosis instead of a chromosome out of two chromatids and one centromer. The consequence is a second fraction during anaphase where the second round of the cycle starts.

B. McCLINTOCK recognized that the fraction cannot occur at any site of the chromosome but is restricted to certain sections that she called Ds (dissociation). These were obviously DNA segments contributing to the formation of translocations, deletions, inversions and to the generation of ring-shaped chromosomes. The first fraction causes similar fractions in the mitosis cycles of following generations. They happen during ontogenesis at different times and sites.

The segment Ds, a mutator gene, behaves like a multiple allele (or, even better, like a pseudoallele) that can be located at different gene loci. It may also vary in structure. This mutator can insert itself into other genes thus rendering them inactive. It is a control element that changes its place within the chromosome, jumping or wandering around and causing mutations wherever it inserts (the mutators are also called jumping genes).

It soon became clear that a further set of elements has to exist: the Ac (activation) elements. A chromosomal fraction or a translocation of a Ds element has to be supported by an Ac element. An Ac element can also be regarded as a multiple allele. It may occur at the most different sites in all chromosomes. To analyze its effect further concentrated B. McCLINTOCK on the study of genes that determine the colour of maize grains.

One of the most important is the C-locus that causes a dark red staining of the aleuron layer and the pericarp of the maize grain in a dominant condition. If a Ds-element jumps into the gene, colour synthesis is interrupted and colourless (yellow) grains result. An Ac activity within these grains causes a pattern of dark red areas on a light ground. This is explained by a reestablishment of the old state since the Ac element removes the Ds from the C-locus. This happens in several cells during the development of the maize grain. These cells are the origin of the aleuron layer and the pericarp and the back mutation can only be perceived in the clones that form out of the changed cells.

Today are a number of gene loci known that can be influenced by the Ds-Ac-system or other control elements. The detection of the spm-system (suppressor-mutator) and the elucidation of its function showed that the control elements do not only act as switches (a yes/ no decision) but that they do modulate the degree of gene expression, too

The genetic analyses of B. McCLINTOCK were not understood for years. Only when insertion elements and transposons were found in bacterial DNA during the late sixties did an analogy between them and the control elements show up. These genetic data fitted neatly with molecular biological models (B. NEVERS and H. SAEDLER, 1977, H-P. DÖRING and P. STARLINGER, 1984). Mrs BARBARA McCLINTOCK was awarded the Nobel prize for medicine and physiology for her pioneer achievements. P.NEVERS, N. S. SHEPHERD and H. SAEDLER listed the 'unstable plant genes' described in literature at the beginning of 1986. It shows that such genes have been found in more than 30 species. Many of the respective mutants with names like variegate, marmorata, maculata or variabilis are on the market as ornamental plants due to their irregularly spotted flowers or leaves.

Holliday junction: central intermediate of genetic recombination - DNA replication with 100% fidelity is a nice feature to keep offspring in just the genetic background of the species. But to get there, or to evolve further, requires genetic changes, one of which results from recombination of (near) homologous parts of DNA. The nature of structural changes in DNA neccessary to result in homologous genetic recombination were layed out by Holliday in 1964, and in subsequent years the crossover-structures were visualized by electron microscopy. The actual conformation of a DNA crossover was speculated to be a four-way-junction with separate DNA helices, or with stacked helices in either a parallel or an antiparallel orientation of the helices. The models had to allow for branch migration, else no exchange of genetic material would happen.

During branch migration hydrogen bonds between paired bases have to be broken and others reformed instead. On average the energy for braking and reforming these bonds will cancel each other - but in real existing DNA not all base pairs are created equal. This calls for the action of enzymes to overcome the neccessary activation energy. And enzymes are needed anyway to resolve the four-way-junctions into separate helices. In E. coli. e.g. there exists an enzyme system (RuvABC) the components of which hold the Holliday junction (RuvA), swivel the DNA strands to enable branch migration (RuvB) and finally cut the junction (RuvC). A DNA ligase restores intact double helices.

Homologous genetic recombination is a highly dynamic process, in contrast to X-ray crystallography relaying on static structures. So it took to the end of the previous millenium to get an atomic detail view of relevant structures. You may see here the structure of a four-way Holliday-junction formed by homologous DNA strands, a RuvA-tetramer complexed to a static Holliday-junction, the motor driving branch migration, and a Holliday-junction resolving enzyme.

The Ruv-System of E. coli is in itself a dynamic complex. During branch migration two tetramers of RuvA hover on both sides of a cruciform DNA, with multimeric RuvB clamping two of the DNA strands to wind them. This complex is not accessible for RuvC. In order for the resolvase to act, one of the RuvA tetramers has to be dissociated so that one side of the DNA junction is amenable to strand separation. In vitro the tetramer-octamer-equilibrium is subject to the salt concentration of the buffer. Conditions neccessary for crystallisation of the complex resulted in tetrameric RuvA complexed to the DNA.

Our research is focused on the molecular mechanisms of genetic recombination, with the long-term objective being the reconstitution of in vitro systems that accurately reproduce the cellular processes. We are characterizing the biochemical properties of proteins essential to homologous recombination, in prokaryotes, eukaryotes, and Archaea.

 In E. coli, the RecA, RecBCD, RecQ, RuvABC, and SSB proteins, and a specific DNA sequence called Chi, are essential to homologous recombination. The RecA protein possesses the unique ability to pair homologous DNA molecules (Figure 3.4) and to promote the subsequent exchange of DNA strands. Since RecA protein is the prototypic DNA strand exchange protein, we are interested in the biochemical mechanism of protein-mediated recognition and exchange of homologous DNA strands. The RecBCD enzyme is both a DNA helicase and a nuclease with the remarkable properties that its nuclease activity, but not its helicase activity, is attenuated by interaction with the Chi sequence, and that it will actively load RecA protein onto ssDNA. RecQ protein is a helicase that can also effect recombination events. SSB protein is an ssDNA binding protein that stimulates the activities of RecA, RecBCD, and RecQ proteins by virtue of its ability to bind ssDNA. Recently, we reconstituted an in vitro pairing reaction that requires the concerted action of each of these proteins; the role of each protein in this reaction is under investigation.

 Figure 3.4: RecA protein-dsDNA complex imaged by atomic force microscopy (AFM):

We are also studying the biochemistry of homologous recombination in the yeast, S. cerevisiae and the archaeon, S. solfataricus. Rad51 and RadA proteins are the RecA protein homologues, respectively. In yeast, at least three ancillary proteins are needed for Rad51 protein-mediated DNA strand exchange: these include the RP-A, Rad52, and Rad54 proteins. We are studying the mechanism of these reconstituted reactions

General Recombination Is Guided by Base-pairing Interactions Between Complementary Strands of Two Homologous DNA Molecules - General recombination involves DNA strand-exchange intermediates that require some effort to understand. Although the exact pathway followed is likely to be different in different organisms, detailed genetic analyses of viruses, bacteria, and fungi suggest that the major outcome of general recombination is always the same

(1) Two homologous DNA molecules "cross over"; that is, their double helices break and the two broken ends join to their opposite partners to re-form two intact double helices, each composed of parts of the two initial DNA molecules (Figure 3.5).

Figure 3.5: General recombination. The breaking and rejoining of two homologous DNA double helices creates two DNA molecules that have "crossed over.”

(2) The site of exchange (that is, where a red double helix is joined to a green double helix (in Figure 3.5) can occur anywhere in the homologous nucleotide sequences of the two participating DNA molecules.

(3) At the site of exchange, a strand of one DNA molecule becomes base-paired to a strand of the second DNA molecule to create a staggered joint (usually called a heteroduplex joint) between the two double helices (Figure 3.6). The hetero-duplex region can be thousands of base pairs long; we shall explain later how it forms.

Figure 3.6: A heteroduplex joint. This structure unites two DNA molecules where they have crossed over. Such a joint is often thousands of nucleotides long 

(4) No nucleotide sequences are altered at the site of exchange; the cleavage and re-joining events occur so precisely that not a single nucleotide is lost or gained. Despite this precision, general recombination creates DNA molecules of novel sequ-ence: the heteroduplex joint can contain a small number of mismatched base pairs, and, more important, the two DNAs that cross over are usually not exactly the same on either side of the joint.

The mechanism of general recombination ensures that two regions of DNA double helix undergo an exchange reaction only if they have extensive sequence homology. The formation of a heteroduplex joint requires that such homology be present because it involves a long region of complementary base-pairing between a strand from one of the two original double helices and a complementary strand from the other. But how does this heteroduplex joint arise, and how do the two homologous regions of DNA at the site of crossing-over recognize each other? As we shall see, recognition takes place by means of a direct base-pairing interaction. The formation of base pairs between complementary strands from the two DNA molecules then guides the general recombination process, allowing it to occur only between long regions of matching DNA sequence.

General Recombination Can Be Initiated at a Nick in One Strand of a DNA Double Helix - Each of the two strands in a DNA molecule is helically wound around the other. As a result, extensive base-pair interactions can occur between two homologous DNA double helices only if a nick is first made in a strand of one of them, freeing that strand for the unwinding and rewinding events required to form a heteroduplex with another DNA molecule. For the same reason, any exchange of strands between two DNA double helices requires at least two nicks, one in a strand of each interacting double helix. Finally, to produce the heteroduplex joint illustrated in Figure 3.6, each of the four strands present must be cut to allow each to be joined to a different partner.

In general recombination, these nicking and resealing events are coordinated so that they occur only when two DNA helices share an extensive region of matching DNA sequence. There is evidence from a number of sources that a single nick in only one strand of a DNA molecule is sufficient to initiate general recombination. Chemical agents or types of irradiation that introduce single strand nicks, for example, will trigger a genetic

recombination event. Moreover, one of the special proteins required for general recombination in E. coli the RecBCD protein has been shown to make single strand nicks in DNA molecules. The RecBCD protein is also a DNA helicase, hydrolyzing ATP and traveling along a DNA helix transiently exposing its strands. By combining its nuclease and helicase activities, the RecBCD protein will create a single-stranded "whisker" on the DNA double helix (Figure 3.7). Figure 3.8 shows how such a whisker could initiate a base-pairing interaction between two complementary stretches of DNA double helix.

 

Figure 3.7: One way to start a recombination event. The RecBCD protein is an enzyme required for general genetic recombination in E. coli. The protein enters the DNA from one end of the double helix and then uses energy derived from the hydrolysis of bound ATP molecules to propel itself in one direction along the DNA at a rate of about 300 nucleotides per second. A special recognition site (a DNA sequence of eight nucleotides scattered throughout the E. coli chromosome) is cut in the traveling loop of DNA created by the RecBCD protein, and thereafter a single-stranded whisker is displaced from the helix, as shown. This whisker is thought to initiate genetic recombination by pairing with a homologous helix, as in Figure 3.8.

Figure 3.8: The initial strand exchange in general recombination. A nick in a single DNA strand frees the strand, which then invades a homologous DNA double helix to form a short pairing region with one of the strands in the second helix. Only two DNA molecules that are complementary in nucleotide sequence can base-pair in this way and thereby initiate a general recombination event. All of the steps shown here can be catalyzed by known enzymes (see Figures 3.7 and 3.11).

DNA Hybridization Reactions Provide a Simple Model for the Base-pairing Step in General Recombination - In its simplest form, the type of base-pairing interaction central to general recombination can be mimicked in a test tube by allowing a DNA double helix to re-form from its separated single strands. This process, called DNA renaturation or hybridization, occurs when a rare random collision juxtaposes complementary nucleotide sequences on two matching DNA single strands, allowing the formation of a short stretch of double helix between them. This relatively slow helix nucleation step is followed by a very rapid "zippering" step as the region of double helix is extended to maximize the number of base-pairing interactions (Figure 3.9).

Figure 3.9: DNA hybridization. DNA double helices re-form from their separated strands in a reaction that depends on the random collision of two complementary strands. Most such collisions are not productive, as shown at the left, but a few result in a short region where complementary base pairs have formed (helix nucleation). A rapid zippering then leads to the formation of a complete double helix. A DNA strand can use this trial-and-error process to find its complementary partner in the midst of millions of non-matching DNA strands. Trial-and-error recognition of a complementary partner DNA sequence appears to initiate all general recombination events. 

Formation of a new double helix in this way requires that the annealing strands be in an open, unfolded conformation. For this reason in vitro hybridization reactions are carried out at high temperature or in the presence of an organic solvent such as formamide; these conditions "melt out" the short hairpin helices formed where base-pairing interactions occur within a single strand that folds back on itself. Bacterial cells could not survive such harsh conditions and instead use a single-strand binding protein, the SSB protein, to open their helices. This protein is essential for DNA replication as well as for general recombination in E. coli; it binds tightly and cooperatively to the sugar-phosphate backbone of all single-stranded regions of DNA, holding them in an extended conformation with their bases exposed. In this extended conformation a DNA single strand can base-pair efficiently with either a nucleoside triphosphate molecule (in DNA replication) or a complementary section of another DNA single strand (in genetic recombination). When hybridization reactions are carried out in vitro under conditions that mimic those inside a cell, the SSB protein speeds up the rate of DNA helix nucleation and thereby the overall rate of strand annealing by a factor of more than 1000.