Assigned Reading: This syllabus. Important terms are bolded. Illustrative and supplementary information (rare) is italicised.

Transcription

1 RECOMBINATION AND GENOME REARRANGEMENTS Date: August 19, 2005 * Time: 1:00pam- 1:50 pm * Room: G-202 Biomolecular Building Lecturer: Dale Ramsden Lineberger dale_ramsden@med.unc.edu *Please consult the online schedule for this course for the definitive date and time for this lecture. Office Hours: by appointment Assigned Reading: This syllabus. Important terms are bolded. Illustrative and supplementary information (rare) is italicised. Basic Principles: For those of you with little background in molecular biology, a review of basic principles of nucleic acid structure and metabolism will be presented (GUTS, or Get Up to Speed session) immediately before this lecture, on August 15 between 9:00 and 9:50 PM Overall objectives for The genome, and genomic instability Genome Maintenance I: Genes and Chromosomes: August 15th Genome Maintenance II: Genome Replication: August 17th Genomic Instability I: DNA damage and repair: August 18th Genomic Instability II: : August 19th This section consists of four lectures: In the the first two you will learn how to define the genome, and describe how genomes are maintained. In the second two lectures, you will learn why genomes are unstable, the consequences of genomic instability, and how cells cells mitigate the risk of genomic instability. Lecture objectives: At the end of this lecture, you should have an idea a) How double strand breaks are repaired, and the consequences of repairing double strand breaks different ways. b) How retroviruses and other mobile elements work c) Why you do V(D)J recombination, and how V(D)J recombination relates to both mobile DNA elements and double strand break repair

2 I. GENERAL CONSIDERATIONS and rearrangements represent a gross change in the organization of your genome. For example, recall you have one maternal and one paternal copy (homologous copies) of each chromosome; During meiotic recombination, paternal and maternal information is mixed up (see e.g. crossover in figure below). However, there also more severe rearrangements, including exchange of material between chromosomes that are NOT homologous (see e.g. translocation between Ch 14 and Ch18 in figure below). Duplications/amplifications, insertions, deletions, and fusions can also occur. Such rearrangements are typically the consequence of DNA double strand break (DSB) repair. Figure 1. Examples of genome rearrangements Sources of DSBs that generate genome rearrangements 1) DSBs can be intentionally made by the cell, as the resulting recombination is required for certain NORMAL developmental programs; meiosis, V(D)J recombination (rearrangement is a good thing) 2) DSBs from exogenous DNA damaging agents (e.g. ionizing radiation, ROS, chemotherapy) are typically repaired so that rearrangements do NOT occur. However, aberrant repair of these DSBs can lead to rearrangements that cause cell death, or cancer (rearrangement is a bad thing) 3) Integrations and excisions of mobile DNA elements (including transposition, retrovirus integrations; see example below) are another source of genome rearrangements. Certain rearrangements (translocations/deletions/insertions) often cause cell death and developmental problems, and are seen in many (most?) cancers - 2 -

3 II. DSB REPAIR A. Homologous recombination REVIEW Chromosomes are found in pairs as homologues (one homologue inherited from mom, one homologue from pop), both of which are duplicated prior to each cell division. The recently replicated copy is termed a sister chromatid. Homologous chromosomes are similar, but because they are derived from different parents, they are not identical. Sister chromatids are made by replication in S phase of every cell cycle, and are identical (with the exception of errors made during replication). How it works Figure 2. Homologous recombination 1) make a double strand break 2) At the region of the break, align the equivalent unbroken region from either the intact homologous chromosome, or the intact sister chromatid 3) use the intact homologue or sister chromatid as a template for repair (yes, its true; the term homologous recombination isnt great). The choice of homologue or sister is made according to cell type. In Meiotic cells: Programmed rearrangement for genetic variability Genetic variability is derived from recombination during meiosis. Meiosis is the process required to make the haploid (one copy of each chromosome) cell (germ cell in multicellular eukaryotes; sperm or oocyte) that is the precursor to sexual reproduction. A diploid progenitor (parent cell) undergoes a round of replication and produces sister chromatids, then undergoes homologous recombination, then divides twice without replication, producing four daughter cells with a haploid DNA content. Each of the four haploid germ cells now contains half the number of chromosomes as the parent cell; also, each chromosome in a given haploid cell is now a unique mixture of the maternal and paternal version of that chromosome. What does meiotic recombination have to do with double strand break repair? between two homologous chromosomes requires one of the chromosomes to be broken. Meiotic cells are programmed to do introduce a double strand break approximately at random in each chromosome. They have an enzyme to do this (expressed only in meiotic cells, called spo11) that looks like a type II topoisomerase, except after making the double strand break, it doesn t reseal it. The intact homologue is then used as a template for repair. occurs between homologues In meiosis un-productive recombination with sister chromatids is blocked; such recombinations would be un-productive because sisters are identical, thus no variability would result - 3 -

4 between homologous chromosomes generates genetic variability in two ways. Gene conversion. Short patch of information around break is converted to be like the unbroken. i.e. if paternal chromosome has double strand break, the region around break is converted to maternal information after repair is finished. Non-reciprocal (I.e maternal copy stays the same). Cross-over. Two chromosomes exchange arms as a consequence of repair. Figure 3. Meiotic recombination WHY homologous recombination can result in gene conversion or crossover is a consequence of how the mechanism works; for details regarding this, see In Mitotic cells: DNA Repair In addition to its role in meiosis, homologous recombination is a critical pathway for repair of double strand breaks made accidentially (e.g. replication through unrepaired strand breaks, or exogenous agents like ionizing radiation). Homologous recombination may also be used for repair of other types of DNA damage. Hereditary breast cancer is caused in part by defects in a gene required for recombinational repair (BRCA2). In mitotic cells, occurs between sister chromatids When recombination is not recombination. in cells undergoing mitosis is skewed such that the intact sister chromatid is used as a template for repair, rather than a homologuous chromosome (opposite to meiotic recombination). This is significant, as sister chromatids are identical (not homologous). Exchange of material between sister chromatids is thus normally conservative, i.e. will not result in a rearrangement or recombination from a genetic standpoint. This ensures the genetic makeup of daughter cells is the same as the parent before recombination. This is why it makes for a good repair pathway in mitotic cells, and why you avoid repair by the sister chromatid in meiotic cells. Nevertheless, the mechanism by which sister chromatids exchange material is essentially the same as homologous recombination, so the term homologous recombination is used to refer to the proccess in general terms, identifying the paired molecules as sisters or homologues when necessary. When recombination is still recombination. Alignment of chromatids is based on homology/identity. Therefore, chromatids can be misaligned when there is homology between two regions on the same chromosome. This is not uncommon, due to frequent repetitive DNA - 4 -

5 elements in the genome (see Genes and chromosomes, and also below) (e.g. Alu elements). and crossover between misaligned chromatids can result in unequal sister chromatid exchange; the products are a duplication of the region between repetitive elements in one sister chromatid, and deletion in the other. B. End joining Figure 4. Unequal sister chromatid exchange Figure 5. End joining. End-joining refers to a pathway for repair of double strand breaks in which broken DNA ends are simply joined back together. How it works Pretty simple, really. Get rid of damage at ends (using nucleases), make the ends compatable (polymerases) then stick whatever is left back together with a ligase. Note that repair junctions almost always involve loss of sequence, thus this pathway is innaccurate

6 Given that end joining is less accurate, why would you choose to do it over homologous recombination? Essentially, End joining is more flexible and easier to do. Homologous recombination End joining Accuracy Conservative Loss of information at ends, prone to translocation Efficiency Requires lots of synthesis, Easy, efficient chromatid/chromosome synapsis Flexibility Restricted to S phase (cells must be actively cycling) Anytime (can occur in resting or differentiated cells) III. MOBILE ELEMENTS A. Transposition Structure Figure 6. A transposon Transposons are mobile elements within a host genome that are able to directly transport themselves to other locations within the genome, or sometimes to another host s genome. The DNA excises from one location, and then integrates into another location. Transposition is a common way for bacteria to acquire resistance to antibiotics. Each transposon contains a gene (or genes) encoding a transposase, the enzyme involved in insertion of the transposon to a new site. Transposons have two signals, which usually flank the genes necessary for transposition. Mobility Excision is targeted by signals. However, the target site for integration is typically chosen with much less specificity. Excision from the host genome is initiated by transposase-mediated strand breaks at the border between signal and host DNA, resulting in a free linear transposon DNA segment, and a double strand break in the host genome at the site of excision. These breaks are dangerous, and unless accurately repaired by the host s double strand break repair machinery can lead to death or recombination Figure 7. Transposon mobility

7 Integration occurs by the introduction of a double strand break at a new target site in the host genome. The linear transposon DNA is inserted by joining the ends of the transposon to the ends of the broken target site. For these reasons, transposons are frequently harmful to the host. After excision, the double strand breaks left behind can lead to cell death, or cancer-causing recombinations. They also can integrate such that they disrupt nearby genes or the expression of nearby genes. B. Retro elements Retro-element life cycle Retro elements are a general term for mobile elements that have an obligatory RNA intermediate. They integrate into host genomes by the same mechanism as transposons do. However, the integrated retro element is NOT excised. Instead, the retro element s genome is transcribed by host transcription machinery as directed by DNA sequences at the 5 end of the element s genome. This RNA copy of the genome is then converted into a DNA copy by the process of reverse Pro ces s Repl ic ation Tran script ion Rever se transc ri ption Enyz me DNA po lymerase RN A po lymerase Rever se transc riptas e Tem plate DNA DNA RNA Produ ct DNA RN A DNA transcription. The new DNA copy can now integrate elsewhere. Reverse transcription represents an important reversal of the normal flow of information in molecular biology (see table). Retro elements can be further divided into 1) Retroviral elements: their genome can be packaged into infectious virus, and thus have an extracellular phase in their life cycle, and 2) Retroposons, whose activity are limited to intracellular events. Like most higher eukaryotes, we have a REDICULOUS number of MOSTLY inactive retroposons already resident in our genome; about 1/4 of our genomic DNA comes from retroposons (remember only about 1/20 th actually codes for genes). They do move occasionally. Insertional mutagenesis As previously mentioned for transposons, insertion of retroelements can disrupt genes. A significant number of congenital, disease-causing mutations are due to insertion of a retroposon into an important gene in germ cells. With respect to retroviruses, the risk of disruption is even Figure 8. Retroviruses vs. Retrotransposons - 7 -

8 greater, as they have a strong preference for integration into or near active genes (likely to a preference for euchromatin). Moreover, the integrated viral genome has strong transcriptional enhancers, thus even the insertion of a retrovirus nearby a gene can result in over expression or anomalous expression of that gene.(see also case conference I) Pathogenic Retroviruses In addition to accidental interference with host cell growth due to its integration, some retroviruses specifically carry genes to disrupt normal cell growth. Retroviral transduction of host sequences and non-autonomous viruses The retrovirus can also acquire growth disrupting capability by recombination between the integrated retrovirus (provirus) and the host genome, or aberrant splicing of the viral RNA to a host gene. This produces a hybrid viral genome, with host sequence partly substituted for part of the viral sequence. The hybrid RNA can contain information necessary for packaging and integration, as well as the host gene, but it usually cannot produce infectious virus on its own because it lacks all the necessary viral genes. This partially defective virus is not active on its own (non-autonomous). However, missing functions can be provided in trans, by an intact helper virus present in the same cell. In some cases the encoded host/viral hybrid protein alone is sufficient to cause cancer. For example, v-src was is an aberrant version of a normal host gene carried by the Rous sarcoma retrovirus, and causes cancer in chickens. In a similar manner, retroviruses can be engineered for use in gene therapy, to deliver genes to cells that are deficient in that gene (see case conference I). IV. V(D)J RECOMBINATION A. Generation of diversity Figure 9. Non-autonomous retroelements. Vertebrates (including us) possess an adaptive immune system. We are able to mobilize subsets of cells that can specifically respond to any one of a virtually infinite variety of different foreign agents, and protect us against them. This ability rests in the diversity present in a class of receptor proteins (e.g. in B cells, the antibody or immunoglobulin) found on the surface of the lymphocytes (white blood cells). As lymphocytes mature, they each generate a different receptor, each with a different specificity. Lymphocytes shown to be useful in recognizing foreign pathogen are activated, and now help clear the body of pathogen. In the spleen and lymph nodes, lymphocytes with specific receptors that have proven useful in this way are then saved for future use (immunological memory, the reason why vaccines work), while the rest are eliminated.this process goes on for the life of the organism

9 Figure 10. Generation of receptor diversity Diversity in antibodies is primarily derived through V(D)J recombination. Multiple versions of coding segments (V, D, or J segments) are distributed at a specific locus in the chromosome, as shown above. One each of a V, D, and J segment is then assembled to code for the complete variable domain of the mature receptor. The variable domain provides specificity for different foreign agents. The remaining domains (constant domains) mediate the immune response to the foreign agent, and are thus common to all the different antibodies. The assembly process, termed V(D)J recombination, occurs at the DNA level and only in developing lymphocytes. One of the loci coding for human antibodies has an estimated 300 different Vs, 12 different Ds, and 4 different Js. This leads to a potential 300X12X4 or over 14,000 different combinations. The junctions of these recombinations are also imprecise, further increasing diversity. Estimates suggest over 100 million different receptors are possible, a number that likely exceeds the pool of lymphocytes we have available at any given time. B. How it works V(D)J recombination occurs in two steps: a lymphocyte specific initiation step (cleavage), and a resolution step (joining) that can occur in any cell type. Initiation: cleavage makes a pair of double strand breaks RAG1 and RAG2 ( Activating Genes 1 and 2) recognize a pair of recombination signals and introduce double strand breaks precisely at the borders between signals and coding segments. RAG1/RAG2 are expressed only in developing B/T cells. Figure 11. V(D)J recombination

10 Resolution: End joining Resolution of these double strand breaks does not require lymphocyte specific genes; rather, resolution requires genes encoding end-joining double strand break repair factors, which are expressed in all cell types. Mutations in factors required for end joining repair leads to both immunodeficiency (because lymphocytes cannot resolve double strand break intermediates in V(D)J recombination) as well as general cellular sensitivity to other sources of double strand breaks (e.g. ionizing radiation sensitivity, because they cannot efficiently repair other sources of double strand breaks either). The ends of coding segment DNA are joined together by the end joining pathway for repair of double strand breaks, forming the variable domain of the antibody. Signal ends are typically also joined together to make a signal junction product. This circular DNA has been removed from its normal chromosomal context, and, under most circumstances, is lost in subsequent cell divisions. An adapted transposon V(D)J recombination possesses many mechanistic similarities to transposition. 1) a pair of signals are used to target a pair of double strand breaks, resulting in the excision of the intervening DNA from the host genome. 2) After excision, the break in the flanking host DNA must be rejoined by host repair machinery. However, we appear to have mostly re-trained this transposon so that the excised DNA is formed into a circular deletion product, rather than integrating elsewhere. C. V(D)J recombination and disease? Severe combined immunodeficiency (scid) A failure in the cleavage step (RAGs) results in complete deficiency in both B and T cells (scid) because they cannot initiate V(D)J recombination (no cleavage). However, some people have radio-sensitive scid, or rs-scid; in addition to no B and T cells, these patients have a general cellular sensitivity to double strand break inducing agents (e.g. ionizing radiation). These people have a defect in a gene required for end joining. Lymphoid malignancy Lymphoid malignancies (leukemia, lymphoma, thymoma) are common. They are often due to translocations, and these translocations frequently have breakpoints in antibody T cell receptor loci near where double strand break intermediates in V(D)J recombination occur

11 Figure 8 reproduced with permission from Genes VII, by B. Lewin, Oxford University Press, 2000, p Figure 9 reproduced with permission from Genes VII, by B. Lewin, Oxford University Press, 2000, p