Chapter 28 DNA Metabolism: Replication, Recombination, and Repair PDF

Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett Chapter 28 DNA Metabolism: Replication, Recombination, and Repair © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distribute...

Reginald H. Garrett Charles M. Grisham www.cengage.com/chemistry/garrett Chapter 28 DNA Metabolism: Replication, Recombination, and Repair © 2017 Cengage Learning. All Rights Reserved. May not be copied, scanned, or duplicated, in whole or in part, except for use as permitted in a license distributed with a certain product or service or otherwise on a password-protected website for classroom use. Chapter 28 Heredity “I am the family face Flesh perishes, I live on, Projecting trait and trace Through time to times anon And leaping from place to place over oblivion.” Thomas Hardy An idealized image of DNA, the substance of heredity. Essential Questions How is genetic information in the form of DNA replicated? How is the information rearranged? How is its integrity maintained in the face of damage? Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? The Dawn of Molecular Biology April 25, 1953 Watson and Crick: "It has not escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." The mechanism: Strand separation, followed by copying of each strand. Each separated strand acts as a template for the synthesis of a new complementary strand. DNA Replication Mindtap DNA replication is semiconservative – one of the two original strands is conserved in each progeny molecule Figure 28.1 DNA replication: Strand separation followed by the copying of each strand. Features of DNA Replication Mostly in E. coli, but many features are general DNA replication is bidirectional o Bidirectional replication involves two replication forks, which move in opposite directions o Supercoiling must be overcome - by DNA helicases and gyrases Figure 28.2 Bidirectional Replication Comparison of labeling pattern expected during unidirectional versus bidirectional replication. An autoradiogram of E. coli chromosome replication. Features of DNA Replication, Con’t Replication is semidiscontinuous o Leading strand is formed continuously o Lagging strand is formed from Okazaki fragments - discovered by Tuneko and Reiji Okazaki – See Figure 28.3 Figure 28.3 The Semidiscontinuous Model for DNA Replication (a) Leading and lagging strand synthesis. Mindtap Newly synthesized DNA is red. (b) The action of DNA polymerase. Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.2 What Are the Functions of DNA Polymerases? The enzymes that replicate DNA are called DNA polymerases All DNA polymerases share the same fundamental catalytic mechanism o The incoming base is selected within the polymerase active site, as determined by Watson-Crick geometric interactions o Chain growth is in the 5′-3′ direction and is antiparallel to the template strand o DNA polymerases cannot initiate DNA synthesis de novo – all require a primer oligonucleotide with a free 3′OH to build upon Biochemical Characterization of DNA Polymerases Watson and Crick predicted the existence of an enzyme that makes DNA copies from a DNA template In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA polymerase - DNA polymerase I (Pol I) – in E. coli DNA polymerases need all four deoxynucleotides, a template, and a primer - a single-stranded DNA/RNA (with a free 3'-OH) that pairs with the template to form a short double-stranded region The new chain is elongated in the 5′→3′ direction, forming a polynucleotide sequence that is antiparallel and complementary to the template The Chain Elongation Reaction Catalyzed by DNA Polymerase The 3'-OH carries out a nucleophilic attack on the αphosphoryl group of the incoming dNTP. PPi is released as a product. The subsequent hydrolysis of PPi by inorganic pyrophosphatase renders the reaction effectively irreversible. E. coli Cells Have Several Different DNA Polymerases The polymerases of E. coli are compared in Table 28.1 Polymerases I, II, and V function principally in DNA repair DNA polymerase III is the chief DNA-replicating enzyme of E. coli There are only 40 molecules of Pol III in an E. coli cell Properties of the DNA Polymerases DNA Pol I was discovered in 1957 by Arthur Kornberg and his colleagues. Pol I and Pol II are involved in DNA repair. Pol III is the enzyme responsible for replication of the E. coli chromosome. DNA Polymerase III The polymerase that carries out replication in E. coli At least 10 different subunits "Core" enzyme has three subunits - α, ε, and θ Alpha (α) subunit is the polymerase Epsilon (ε) subunit is a 3'-exonuclease Theta (θ) subunit is involved in holoenzyme assembly and ε-subunit stabilization DNA Polymerase III The β subunit dimer “sliding clamp” forms a ring around DNA Enormous processivity - 5 million bases! DNA Polymerase III The Pol III holoenzyme consists of 17 subunits, (αεθ)22β2τ2γδδ′χψ This is the form that carries out replication o The γ-complex (τ2γδδ′χψ) is responsible for assembly of the DNA polymerase III holoenzyme complex The γ-complex of the holoenzyme acts as a clamp loader by catalyzing the ATP-dependent transfer of the β2 sliding clamp to each DNA template Each β2 sliding clamp forms a closed ring around a DNA strand that can slide along the DNA during replication, tethering the core polymerase to the template The Composition of E. coli Pol III Figure 28.5 DNA Polymerase III Holoenzyme Is a Dimeric Polymerase One unit of polymerase synthesizes the leading strand, and the other synthesizes the lagging strand E. Coli Pol III Is a Dimeric Polymerase One unit of polymerase synthesizes the leading strand, and the other synthesizes the lagging strand All template strands are read in the 3'-5' direction, so DNA synthesis proceeds in the 5'-3' direction Lagging strand synthesis requires repeated priming Primase (DnaG) bound to the DnaB helicase carries out this priming function, periodically forming new RNA primers on the lagging strand All single-stranded regions of DNA are coated with SSB (single-stranded DNA-binding protein) A Pol III Holoenzyme Sits at Each Replication Fork The features of the replication fork are shown in Figure 28.7 DNA gyrase (topoisomerase) and DnaB helicase unwind the DNA double helix The lagging strand is looped around, and each replicative DNA polymerase moves 5′→3′ relative to its strand, copying template and synthesizing a new DNA strand More Features of Replication Each replicative polymerase is tethered to the DNA by its β2 sliding clamp Downstream on the lagging strand, DNA pol I excises the primer and replaces it with DNA DNA ligase seals the "nicks" between Okazaki fragments See Figures 28.3 and 28.7 for a view of replication fork General Features of a Replication Fork Figure 28.7 General features of a replication fork. The DNA duplex is unwound by the action of DNA gyrase and helicase, and the separated single strands are coated with SSB. Primase periodically primes synthesis on the lagging strand. Each half of the dimeric replicative polymerase is a “core” polymerase bound to its template strand by a β-subunit sliding clamp. DNA Replication in E. coli Requires a Family of Proteins Properties of E. coli DNA Polymerase I Replication occurs 5' to 3' Nucleotides are added at the 3'-end of the strand Pol I catalyzes about 20 cycles of polymerization before it dissociates from template 20 cycles constitutes moderate "processivity" Pol I from E. coli is a 928-amino acid (109-kD) monomer In addition to 5'-3' polymerase, it also has 3'-5' exonuclease and 5'-3' exonuclease activities Properties of the DNA Polymerases DNA Pol I was discovered in 1957 by Arthur Kornberg and his colleagues. Pol I and Pol II are involved in DNA repair. Pol III is the enzyme responsible for replication of the E. coli chromosome. 3'-Exonuclease Activity of Pol I Removes Nucleotides From the 3'-End of the Chain Why does Pol I have exonuclease activity? The 3'-5' exonuclease activity serves a proofreading function The 3’-exonuclease is a property of the ε-subunit It removes incorrectly matched bases, so that the polymerase can try again The polymerase active site is a proofreader, and the 3’-exonuclease activity is an editor Figure 28.8 The 3’-5’ Exonuclease Activity of DNA Polymerase I The exonuclease removes nucleotides from the 5’-end of the growing DNA chain. The newly synthesized strand is in purple. Properties of the DNA Polymerases DNA Pol I was discovered in 1957 by Arthur Kornberg and his colleagues. Pol I and Pol II are involved in DNA repair. Pol III is the enzyme responsible for replication of the E. coli chromosome. How Does the 5'-Exonuclease Activity of Pol I Accomplish Nick Translation? The 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation" Hans Klenow used trypsin to cleave E. coli pol I between residues 323 and 324, separating 5'exonuclease (on residues 1-323) and the other two activities (on residues 324-928, the so-called "Klenow fragment”) This 5'-exonuclease activity plays an important role in primer removal during DNA replication Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.3 Why Are There So Many DNA Polymerases? Cells have different DNA polymerases for different purposes Polymerases can be grouped in seven functional families, based on sequence homology Family A includes polymerases involved in DNA repair in bacteria Family B includes the eukaryotic polymerases involved in replication of chromosomal DNA 28.3 Why Are There So Many DNA Polymerases? Family C is that of the bacterial chromosomal DNA-replicating enzymes Families X and Y act in DNA repair pathways RT designates retrovirus polymerases Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.4 How Is DNA Replicated in Eukaryotic Cells? DNA replication in eukaryotic cells is similar to that in prokaryotes, but vastly more complex Eukaryotic DNA is organized into chromosomes (with 6 billion base pairs distributed among 46 chromosomes) The cell cycle controls the timing of DNA replication Eukaryotic Cells Contain a Number of Different DNA Polymerases At least 19 different DNA polymerases have been found in eukaryotic cells so far Multiple polymerases participate in leading- and lagging-strand synthesis, especially α, δ, and ε α functions in initiation of nuclear DNA replication Polymerase δ is the principal DNA polymerase in lagging-strand DNA replication; polymerase ε in leading-strand Eukaryotic Cells Contain a Number of Different DNA Polymerases Through its association with PCNA (proliferating cell nuclear antigen) and CMG, polymerases δ and ε carry out highly processive DNA synthesis PCNA and CMG are the eukaryotic counterparts of the E. coli β2-sliding clamp; they clamp δ and ε to the DNA template Biochemical Properties of the Principle Human DNA Polymerases Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.5 How Are the Ends of Chromosomes Replicated? Telomeres are the structures at the ends of eukaryotic chromosomes Telomeres are short (5 to 8 bp) tandemly repeated, G-rich nucleotide sequences that form protective caps 1-12 kbp long on the chromosome ends Vertebrate telomere consensus sequence: TTAGGG Telomerase (an RNA-dependent DNA polymerase) maintains telomere length by restoring telomeres at the 3'-ends of chromosomes 28.5 How Are the Ends of Chromosomes Replicated? Somatic cells, which lack telomerase, inevitably lose bits of their telomeres The telomere theory of aging suggests that cells senesce and die when their telomeres are gone 28.5 How Are the Ends of Chromosomes Replicated? Figure 28.14 Telomere replication. (c) In lagging strand replication, short RNA primers (pink) are added and extended by DNA polymerase. (d) Asterisks indicate telomere sequences. Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.6 How Are RNA Genomes Replicated? Many viruses have genomes composed of RNA DNA is an intermediate in the replication of RNA viruses The viral RNA is the template for DNA synthesis The RNA-directed DNA polymerase is called reverse transcriptase All RNA tumor viruses contain such an enzyme within their viral particle 28.6 How Are RNA Genomes Replicated? RNA viruses that replicate their RNA via a DNA intermediate are termed retroviruses The primer for reverse transcriptase is a specific tRNA molecule captured from the host cell 28.6 How Are RNA Genomes Replicated? Reverse transcriptase transcribes the RNA template into a complementary cDNA strand to form a DNA:RNA hybrid Reverse transcriptase has three enzyme activities: 1) RNA-directed DNA polymerase activity 2) RNase H activity (an exonuclease activity that degrades RNA chains in DNA:RNA hybrids) 3) DNA-directed DNA polymerase activity (which replicates the ssDNA remaining after RNase H degradation of the viral genome), yielding a DNA duplex which directs the remainder of the viral infection process 28.6 How Are RNA Genomes Replicated? HIV reverse transcriptase is of great clinical interest because it is the enzyme for AIDS virus replication DNA synthesis by HIV reverse transcriptase is blocked by nucleotide analogs such as AZT and 3TC HIV reverse transcriptase incorporates these analogs into growing DNA chains in place of dTMP (in the case of AZT) or dCMP (in the case of 3TC) 28.6 How Are RNA Genomes Replicated? Once incorporated, these analogs block further chain elongation because they lack a 3'-OH where the next incoming dNTP can be added The high error rate of HIV reverse transcriptase means that the virus is ever-changing, which makes it difficult to devise an effective vaccine HIV Reverse Transcriptase Inhibitors: AZT Figure 28.15 The structure of AZT (3'-azido-2',3'dideoxythymidine). This nucleoside is phosphorylated in vitro to form deoxynucleoside-5'triphosphate substrate analogs for HIV reverse transcriptase. HIV Reverse Transriptase Inhibitors: 3TC Figure 28.15 The structure of 3TC (2',3'-dideoxy-3'thiacytidine). This nucleoside is phosphorylated in vivo to form deoxynucleoside-5'triphosphate substrate analogs for HIV reverse transcriptase. Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.7 How Is the Genetic Information Shuffled by Genetic Recombination? Genetic recombination rearranges genetic information, creating new associations Recombination involving similar DNA sequences is called homologous recombination Recombination involving very different nucleotide sequences is nonhomologous recombination Transposition is the enzymatic insertion of a transposon, a mobile segment of DNA 28.7 How Is the Genetic Information Shuffled by Genetic Recombination? The process underlying homologous recombination is termed general recombination General recombination requires the breakage and reunion of DNA strands Meselson and Weigle showed that recombination involves exchange of DNA segments Meselson and Weigle’s Experiment Figure 28.16 Meselson and Weigle’s experiment. Density-labeled “heavy” phage (ABC) were used to co-infect bacteria along with “light” abc phage. The progeny from the infection were collected and subjected to CsCl density gradient centrifugation. The progeny included recombinant phage – ABc, Abc, aBc, aBC, and so on, distributed diffusely between the two parental bands (ABC and abc) because they contained chromosomes constituted from fragments of both “heavy” and “light” DNA. Homologous Recombination Proceeds According to the Holliday Model In 1964, Robin Holliday proposed a model for homologous recombination Two homologous DNA duplexes first juxtapose so that their sequences are aligned – a process of chromosome pairing called synapsis Recombination starts with introduction of small nicks at homologous sites on the two chromosomes Homologous Recombination Proceeds According to the Holliday Model Duplexes partially unwind, and the free, singlestranded end of one duplex begins to base-pair with its nearly complementary single-stranded region along the intact strand in the other duplex This process is called strand invasion. Ligation follows, forming a Holliday junction The Holliday Model for Homologous Recombination Figure 28.18 The + and – signs label strands of like polarity. For example, assume that the two strands running 5' to 3' as read are labeled +; and the two strands running 3' to 5' as read left to right are labeled -. Only strands of like polarity exchange DNA during recombination. The Holliday Model for Homologous Recombination Figure 28.18 continued. The + and – signs label strands of like polarity. Nicks take place, either at W and E, or at N and S. Nicks at W and E yield patch recombinant heteroduplexes; nicks at N and S yield splice recombinant heteroduplexes. Transposons Are DNA Sequences That Can Move from Place to Place in the Genome Barbara McClintock first proposed (in 1950) that activator genes could cause mutations in other genes McClintock’s research showed (surprisingly) that activator genes could move about the genome Her “jumping genes” model was viewed with skepticism at first, but molecular biologists verified her model in the 1970s, and in 1983, she was finally awarded the Nobel Prize in Physiology or Medicine for this remarkable discovery McClintock’s jumping genes are now designated as mobile elements, transposable elements, or simply transposons Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.8 Can DNA Be Repaired? A fundamental difference from RNA or protein RNA and protein are replaceable, but DNA must be preserved Cells require a means for repair of: missing, altered, or incorrect bases; bulges due to insertion or deletion; UVinduced pyrimidine dimers; strand breaks; or cross-links The human genome has about 150 genes associated with DNA repair DNA repair systems include: direct reversal damage repair, single-strand damage repair, double-strand break repair, and translesion DNA synthesis 28.8 Can DNA Be Repaired? Enzymatic reactions that reverse the damage, returning DNA to its proper state, are direct reversal repair systems. Single-strand damage repair relies on the intact complementary strand to guide repair Systems repairing single-strand breaks include: o Mismatch repair (MMR) o Base excision repair (BER) o Nucleotide excision repair (NER) Double-strand breaks (DSBs) are a particular threat to genome stability, because lost sequence information cannot be recovered from the same DNA Mismatch Repair Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region, and replace it by DNA polymerase-mediated local replication Methyl-directed pathway (E. coli) is an example Since methylation occurs post-replication, repair proteins identify the methylated strand as parent, remove mismatched bases on the new (unmethylated) strand, and replace them Reversing Chemical Damage Pyrimidine dimers can be repaired by photolyase Base excision repair: DNA glycosylase removes damaged base, creating an apurinic or apyrimidinic "AP” site o AP endonuclease cleaves backbone, exonuclease removes several residues, and gap is repaired by DNA polymerase and DNA ligase Nucleotide excision repair recognizes and repairs larger regions of damaged DNA than base excision repair Repair of Pyrimidine Dimers Formed by UV Light Figure 28.28 UV irradiation causes dimerization of adjacent thymine bases. A cyclobutyl ring is formed between carbons 5 and 6 of the pyrimidine rings. Normal base pairing is disrupted by the presence of such dimers. Photolyase binds at the dimer and uses the energy of visible light to break the cyclobutyl ring, restoring the pyrimidines to their original form. Base Excision Repair Figure 28.29 Base excision repair. A damaged base (black) is excised from the sugarphosphate backbone by DNA glycosylase, creating an AP site. Then, an AP endonuclease severs the DNA strand. (move to the next slide) Base Excision Repair Figure 28.29 continued: Base excision repair. An excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap. Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? 28.9 What Is the Molecular Basis of Mutation? Mutations change the sequence of bases in DNA, either by o Substitution of one base for another (so-called point mutations) o Or by the insertion or deletion of one or more base pairs (insertions or deletions) Mindtap x 2 Point Mutations Arise by Inappropriate Base-Pairing Point mutations arise when a base pairs with an inappropriate partner The two possible kinds of point mutations are: o Transitions (one purine or pyrimidine for another) o Transversions (a pyrimidine for a purine, or vice versa) Bases rarely mispair (Figure 28.30) Mutations can be induced by base analogs o Such as 5-bromouracil (5-BU) o Or 2-aminopurine Point Mutations Due to Base Mispairings Figure 28.30 (a) An example based on tautomeric properties. (b) A in syn conformation pairing with G. (c) T and C base pairing. Mutations Can Be Induced by Base Analogs Figure 28.31 5-Bromouracil usually favors the keto tautomer that mimics the base-pairing properties of thymine, but it frequently shifts to the enol form, whereupon it can base-pair with guanine, causing a T-A to C-G transition. Mutations Can Be Induced by Base Analogs Figure 28.32 (a) 2Aminopurine normally basepairs with T but (b) may also pair with cytosine through a single hydrogen bond. Mutations Can Be Induced by Base Analogs Figure 28.33 Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an A-T to G-C transition. Chemical Mutagens React with the Bases in DNA Figure 28.34 Chemical mutagens. (a) HNO2 (nitrous acid) converts cytosine to uracil and adenine to hypoxanthine. Chemical Mutagens React with the Bases in DNA Figure 28.34 Chemical mutagens. (c) Hydroxylamine (NH2OH) reacts with cytosine, converting it to a derivative that basepairs with adenine instead of guanine. The result is a C-G to T-A transition. Chemical Mutagens React with the Bases in DNA Figure 28.34 Chemical mutagens. (d) Alkylation of G residues to give O6methylguanine, which base-pairs with T. Outline 28.1 How is DNA replicated? 28.2 What are the functions of DNA polymerases? 28.3 Why are there so many DNA polymerases? 28.4 How is DNA replicated in eukaryotic cells? 28.5 How are the ends of chromosomes replicated? 28.6 How are RNA genomes replicated? 28.7 How is the genetic information rearranged by genetic recombination? 28.8 Can DNA be repaired? 28.9 What is the molecular basis of mutation? Special Focus: Gene rearrangements and immunology – is it possible to generate protein diversity using genetic recombination? (SKIP) Do You Understand… 1. DNA replication? 2. Enzymology? 3. DNA repair? 4. Recombination? 5. Alteration in DNA sequence?