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Biochemistry for Pharmacy Students NUCLEIC ACID STRUCTURE & FUNCTION Pál Bauer Dept. of Medical Biochemistry Rm. 4515, EOK Email: [email protected] OBJECTIVES • To learn the structures and properties of nucleotides, the building blocks of nucleic acids • To describe how the structure of DNA relates to functions of the genetic machinery. • To explain how DNA is synthesized. • To describe how mutations in DNA can lead to genetic diseases. • To explain how recombinant DNA technology can be applied to the diagnosis and therapy of human genetic diseases (discussion sessions). HISTORICAL PERSPECTIVE The beginnings of nucleic acid biochemistry & genetics occurred in the 1860s: - Frederic Miescher – Swiss biochemist who discovered nucleic acids. - Gregor Mendel – Austrian monk who founded the science of genetics. Nucleic acid structure & metabolism is directly relevant to cancer, gout, many genetically inherited diseases, AIDS and other infectious diseases. Frederick Meischer studied salmon Gregor Mendel studied garden peas • Sugars and phosphates – Ribose in RNA – Deoxyribose in DNA • Partial hydrolysis products – Nucleotides (contains phosphates) – Nucleosides (no phosphates) Names of nucleosides Base Ribonucleoside Deoxyribonucleoside Adenine Adenosine Deoxyadenosine Guanine Guanosine Deoxyguanosine Cytosine Cytidine Deoxycytidine Uracil Uridine Deoxyuridine Thymine Thymine riboside Thymidine Nucleosides: numbering system • The intra-nucleotide linkage Base composition of double stranded DNA • • • [pyrimidines] = [purines] Content: A = T, G = C DNA base compositions are the same for different tissues of the same organism. Watson-Crick base pairing Crick Watson A Reminder of the Differences between Eukaryotes and Prokaryotes Animal Cell (eukaryote) E. coli Bacterial Cell (prokaryote) Plant Cell Fig. 2-7 (eukaryote) Model of the Nuclear Envelope Artwork by Don Guzy 5’ • The Watson-Crick Structure 3’ • • • • Right-handed helices Anti-parallel Base-pairing Open structure accessible to water • Stacking forces between planar paired bases give a rigid structure 5’ 3’ Denaturation of DNA By pH, heat, solvents, urea, amides • Helix-coil transition • Hyperchromic effect • Melting temperature Denaturation of double-stranded DNA DNA IS THE GENETIC MATERIAL IN CELLS Indirect evidence a. High DNA content of chromosomes b. 260 nm is a very mutagenic wavelength; bases maximally absorb light energy at 260 nm c. Constancy of [DNA] / cell (germ cells) and 2x [DNA] / cell (somatic cells) Direct Evidence a. Transformation of bacteria with DNA Requires both DNA functions: replication and expression b. Transfection with viral nucleic acids c. In vitro expression of DNA (from bacteriophage T4) d. Synthesis of active DNA in vitro Pneumococci DNA CONTENT OF SOME CELLS AND VIRUSES Source of DNA Viruses SV40 Papilloma (wart) Adenoviruses Herpesviruses Poxviruses Haploid size of genome, base pairs____ 5 x 103 (5 kb) 8 x 103 2.1 x 104 1.56 x 105 2.4 x 105 Cells Escherichia coli Yeast Drosophila Human Frogs Onion Plant Fern Plant Animal mitochondria Plant chloroplast 4.5 x 106 1.3 x 107 1.6 x 108 3.2 x 109 6 x 109 18 x 109 160 x 109 (4,500 kb) (3.2x106 kb) (6 x 106 kb) 1.5 x 104 (15 kb) 1 x 105 The total length of the circular E. coli chromosome is 1.7 mm, whereas the length of an E. coli cell is 2 μm. partially lyzed E. coli cell Human haploid genome 22 autosomal chromosomes (chromosomes 1-22) plus X and Y chromosomes 3.2 x 109 total bp • Histones and chromosome structure a. Nucleosomes The doublestranded DNA is wrapped around the outside of each nucleosome twice. The nucleosomes are regularly spaced along the DNA. electron micrograph of chromatin electron micrograph of supercoiled nucleosomes in chromatin. A typical phone cord is coiled like a DNA helix, and the coiled cord can itself coil in in a supercoil. If twisted tight enough, the supercoils will themselves form an even higher order of supercoiling. Double-stranded DNA helices also form supercoils of supercoils. Two drawings depicting the different levels of DNA supercoiling that provide DNA compaction in a eukaryotic chromosome. The levels take the form of coils upon coils. nucleosomes histones DNA replication 3’ 5’ 5’ 5’ 3’ 5’ 3’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ All DNA polymerases catalyze elongation of the primer strand in a 5’ to 3’ direction, copying the template strand in a 3’ to 5’ direction. This means that the “leading strand” can be synthesized continuously, but the “lagging strand” must be synthesized discontinuously. A problem is that DNA polymerases must have a primer strand with a 3’ OH from which to begin DNA synthesis. So, where do these primers come from when a DNA polymerase synthesizes new Okazaki fragments in the lagging strand? Replication does not usually begin at the end of a DNA molecule; it begins in the middle of the DNA. 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ (DNA pol I, II and III) DNA 12.4 Schematic model of the proofreading function of DNA polymerase Figure 12-21 An example of error correction by the 3’ to 5’ exonuclease (proofreading) activity of DNA polymerase I. A mismatched base pair (a C-A mismatch) impedes the movement of DNA polymerase I to the next site. Sliding backward, the enzyme removes its mistake with the 3’ to 5’ exonuclease activity & then resumes its 5’ to 3’ polymerase activity. c. DNA Polymerase II • Mutants in the E. coli gene for this DNA polymerase are not lethal. Also is a repair enzyme. • Requires duplex DNA template and primer. • Utilizes a template strand and elongates a primer strand, similar to DNA polymerase I. d. DNA Polymerase III (pol C gene) Mutants are ts (temperature sensitive), i.e., conditionally lethal. The 3-D structure of E. coli DNA polymerase I. The active site for the polymerase activity and the 3’ to 5’ exonucelase activity is deep in the crevice at the far end of the bound DNA. The template strand is dark blue. a. Illustration of the 5’ to 3’ exonuclease activity of E. coli DNA polymerase I (sometimes called a “nick translation” activity). An RNA or DNA strand paired with a DNA template strand is simultaneously degraded by the 5’ to 3’ exonuclease activity & is replaced by the polymerase activity of the same enzyme. b. The origin of replication – the ori C locus Accessory proteins dna B gene product Probably this protein is membrane-associated and recognizes the initiation sequence on DNA. c. RNA primers DNA polymerases require a preformed primer; RNA polymerases do not. d. Details of the events: 1. Accessory protein binds (dna B protein) 2. "Primase," an RNA polymerase (dna G) It does not require a primer; forms a short piece of RNA complementary to the DNA template strand. 3. DNA binding proteins cell membrane cell membrane 5’ 5’ 3’ 5’ 5’ 3’ 5’ 3’ Additional information about the overall process of DNA replication The 5’ end of RNA primer is usually 5’ pppA... or 5’ pppG…. DNA polymerase III dissociates the primase that synthesizes the RNA primer. The 5' → 3' nuclease of DNA polymerase l, or another enzyme called RNase H, removes the RNA primer. RNase H hydrolyzes RNA in DNA/RNA base-paired regions. DNA polymerase I (and maybe DNA pol II?) fill the gaps left by the removal of the RNA primers. The complete but "nicked" (broken) daughter strands that result after the gaps are filled in are then joined by DNA ligase. • DNA ligase (animal enzyme) • The unwinding problem: 5000 turns/min in E. coli (helicases and topoisomerases) Helicases: unwind the two strands of DNA & cause supercoiling ahead of the fork. Topoisomerase I: relaxes supercoils a. Enzyme recognizes right-handed superhelices introduced during DNA replication. b. Small DNA segment unwinds, as strand breaks, the superhelix relaxes. c. Enzyme, still attached to broken strand, reseals the break. d. The topoisomerase is both a nuclease and a ligase. (The nucleolytic activity can be viewed as the reverse of ligase action.) SUMMARY OF DNA REPLICATION IN BACTERIA Map of the E. coli genome showing some of the genes that encode proteins involved in DNA replication, repair and recombination. DNA REPLICATION in EUKARYOTIC CELLS is SOMEWHAT MORE COMPLICATED and INVOLVES ADDITIONAL DNA POLYMERASES & MULTIPLE REPLICATION ORIGINS Greek Name Gene Name Proposed Main Function alpha POLA DNA Replication beta POLB Base Excision Repair gamma POLG Mitochondrial Replication delta POLD1 DNA Replication epsilon POLE DNA Replication zeta POLZ Bypass Synthesis eta POLH Bypass Synthesis theta POLQ DNA Repair iota POLI Bypass Synthesis kappa POLK Bypass Synthesis lambda POLL Base Excision Repair mu POLM Non-Homologous End Joining sigma POLS Sister Chromatid Cohesion • The number of known eukaryotic DNA polymerases has doubled in the last several years as researchers discover additional enzymes with DNA polymerizing activity. • Most of the newly discovered DNA polymerases are involved in repair of DNA, rather than DNA replication. • The DNA polymerases alpha, delta and epsilon are the most clearly involved in DNA replication. • DNA polymerases delta and epsilon have proof-reading 3’->5’ exonuclease activity; the other DNA polymerases do not. Multiple replication origins occur in the large eukaryotic chromosomes. Mutation Types and rates of mutation Type Genome mutation Mechanism chromosome missegregation (e.g., aneuploidy) Frequency________ 10-2 per cell division Chromosome mutation chromosome rearrangement (e.g., translocation) 6 X 10-4 per cell division Gene mutation base pair mutation 10-10 per base pair per (e.g., point mutation, cell division or or small deletion or 10-5 - 10-6 per locus per insertion generation Mutation rates* of selected genes Gene Achondroplasia Aniridia Duchenne muscular dystrophy Hemophilia A Hemophilia B Neurofibromatosis -1 Polycystic kidney disease Retinoblastoma New mutations per 106 gametes 6 to 40 2.5 to 5 43 to 105 32 to 2 to 44 to 100 60 to 120 5 to 12 57 3 *mutation rates (mutations / locus / generation) can vary from 10-4 to 10-7 depending on gene size and whether there are “hot spots” for mutation (the frequency at most loci is 10-5 to 10-6). Many polymorphisms exist in the genome • the number of existing polymorphisms is ~1 per 500 bp • there are ~5.8 million differences per haploid genome • polymorphisms were caused by mutations over time • polymorphisms called single nucleotide polymorphisms (or SNPs) are being catalogued by the Human Genome Project as an ongoing project Types of base pair mutations normal sequence CATTCACCTGTACCA GTAAGTGGACATGGT transition (T-C to A-G) CATCCACCTGTACCA GTAGGTGGACATGGT transversion (T-A to G-C) CATGCACCTGTACCA GTACGTGGACATGGT base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine deletion CATCACCTGTACCA GTAGTGGACATGGT insertion CATGTCACCTGTACCA GTACAGTGGACATGGT deletions and insertions can involve one or more base pairs Spontaneous mutations can be caused by tautomers Tautomeric forms of the DNA bases Adenine Cytosine AMINO IMINO Tautomeric forms of the DNA bases Guanine Thymine KETO ENOL Mutation caused by tautomer of cytosine Cytosine Normal tautomeric form Guanine Cytosine Rare imino tautomeric form Adenine • cytosine mispairs with adenine resulting in a transition mutation Mutation is perpetuated by replication C G C G • replication of C-G should give daughter strands each with C-G C G C A • tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands C A T A • which could result in a C-G to T-A transition mutation in the next round of replication, or if improperly repaired Chemical mutagens Deamination by nitrous acid O Attack by oxygen free radicals leading to oxidative damage N • many different oxidative modifications occur • by smoking, etc. • 8-oxyG causes G to T transversions NH NH N NH2 guanine O H N NH O NH N NH2 8-oxyguanine (8-oxyG) • the MTH1 protein degrades 8-oxy-dGTP preventing misincorporation • mutation of the MTH1 gene causes increased tumor formation in mice Ames test for mutagen detection • named for Bruce Ames • reversion of histidine mutations by test compounds • His- Salmonella typhimurium cannot grow without histidine • if test compound is mutagenic, reversion to His+ may occur • reversion is correlated with carcinogenicity Thymine dimer formation by UV light Summary of DNA lesions Missing base Acid and heat depurination (~104 purines per day per cell in humans) Altered base Ionizing radiation; alkylating agents Incorrect base Spontaneous deaminations cytosine to uracil adenine to hypoxanthine Deletion-insertion Intercalating reagents (acridines) Dimer formation UV irradiation Strand breaks Ionizing radiation; chemicals (bleomycin) Interstrand cross-links Psoralen derivatives; mitomycin C Tautomer formation Spontaneous and transient Correlation between DNA repair activity in fibroblast cells from various mammalian species and the life span of the organism 100 human elephant Life span cow 10 hamster rat mouse shrew 1 DNA repair activity Defects in DNA repair or replication All are associated with a high frequency of chromosome and gene (base pair) mutations; most are also associated with a predisposition to cancer, particularly leukemias • Xeroderma pigmentosum • caused by mutations in genes involved in nucleotide excision repair • associated with a >1000-fold increase of sunlight-induced skin cancer and with other types of cancer such as melanoma • Ataxia telangiectasia • caused by gene that detects DNA damage • increased risk of X-ray • associated with increased breast cancer in carriers • Fanconi anemia • caused by a gene involved in DNA repair • increased risk of X-ray and sensitivity to sunlight • Bloom syndrome • caused by mutations in a a DNA helicase gene • increased risk of X-ray • sensitivity to sunlight • Cockayne syndrome • caused by a defect in transcription-linked DNA repair • sensitivity to sunlight • Werner’s syndrome • caused by mutations in a DNA helicase gene • premature aging Transition • Transition vs. transversion Exchange of a purine with a purine or pyrimidine with a pyrimindine base. More common than transversion. Often the result of tautomeric shifts. – GC AT transition • causal agents: e.g. base analog 5’bromouracil – AT GC transition • causal agents: e.g. base analog 2-aminopurine • Transversion Exchange of a purine with a pyrimidine base and vice versa – GC TA or GC CG transversion – AT CG or AT TA transversion Methyl-directed mismatch repair 1. Mismatch within 1 kb of 5’ CH3 CH3 MutS, MutL, ATP ADP+Pi 5’ CH3 MutL MutH, ATP ADP+Pi CH3 5’ 3’ 5’ 3’ CH3 MutS 2. MutS and MutH bind to mismatched spots along the DNA (except C-C) 3. DNA on both sides of the Mitsmatch runs through MutS:MutL complex CH3 MutS MutH MutL MutH CH3 methylated GATC 4. MutH binds to MutL and to GATC CH3 5. Endonuclease of MutH cleaves unmethylated DNA at hemimethylated GATC Mismatch repair in E. coli Scenario 1 Mismatch is at the 5’ end of cleavage site 1. Unmethylated DNA is unwound via DNA helicase II 2. The 3’-5’ exonuclease activity of exonuclease I or exo X degrades DNA through the mismatch 5’ 3’ ATP ADP+Pi 5’ 3’ 3. DNA polymerase III synthesizes the new DNA strand 4. DNA ligase closes the remaining nick. CH3 5’ 3’ CH3 CH3 CH3 MutS-MutL DNA helicase II exonuclease I or exonuclease X 3’ 5’ CH3 3’ 5’ DNA polymerase III SSBs CH3 3’ 5’ Mismatch repair in E. coli Scenario 2 Mismatch is at the 3’ end of cleavage site 1. Unmethylated DNA is unwound via DNA helicase II 2. The 5’-3’ exonuclease activity of exonuclease VII or RecJ nuclease degrades DNA through the mismatch 5’ 3’ ATP ADP+Pi 5’ 3’ 3. DNA polymerase III synthesizes the new DNA strand. 4. DNA ligase closes the remaining nick. CH3 5’ 3’ CH3 CH3 CH3 MutS-MutL DNA helicase II exonuclease VII or RecJ nuclease 3’ 5’ CH3 3’ 5’ DNA polymerase III SSBs CH3 3’ 5’ • • • • • Base excision repair not restricted to a short time post replication similar in most organisms (bacteria – mammals) recognizes abnormal bases in the DNA usually less expensive than mismatch repair requires four enzymes 1. 2. 3. 4. DNA glycosylases AP-endonucleases DNA polymerase I DNA ligase • DNA glycosylases G P Relatively small P O enzymes (20 – 30 KDa) • Recognize abnormal bases – deaminated bases – alkylated bases • Remove base via cleavage at the glycosidic bond between the deoxyribose and the base • Cleavage creates apurinic and apyrimidinic (AP sites) U P G O P O O P A O Before P A O After DNA glycosylases Enzyme Uracil DNA glycosylase Hypoxanthin DNA glycosylase 3- methyladenine I DNA glycosylase 3- methyladenine II DNA glycosylase (*) Formamidopyrimidine DNA glycosylase Units / mg protein (x 10 - 3) not adapted 3,800 2.1 3.6 0.22 4.2 adapted 3,500 2 3.6 4.1 3.9 Legend: Adapted = incubation with 1 g/ml NNG for 1 hour Not adapted = untreated control Source: Karran et al. (1983), Nature 296, 770 - 773 AP-endonucleases 5’ P P P P P P P • recognize AP-sites A G G C A G C • cleave phosphodiester bonds near the AP site T C C T C G and generate a 5’ – 3’ P P P P P P P phosphate and 3’AP endonuclease hydroxyl • In E. coli this enzyme also has 3’-5’ exonuclease P P P P P P P activity A G G C A G C • The 3’-OH functions as a T C C G primer 5’ P P 3’ P P Base excision repair (BER) P P P P P P A G G C Uracil DNA glycosylase A T U C G T P P P P P G P P G C C G P P P AP endonuclease P P A P G T G C P P C C P P DNA polymerase I G P DNA ligase P P A P G T P G C G P P • Defects in BER In humans BER involves: – DNA polymerase beta – The Xrcc1 geneproduct – Ape1 (first step in removing the damaged base) • BER deficiencies have been implicated with: – cancer – neurodegenerative diseases – aging • • • • • • Nucleotide excision repair Recognizes large distortions in the DNA structure Repairs UV-damaged DNA Multisubunit enzyme Cleaves two phosphodiester bonds upstream and downstream of the lesion Generally generates fragments of 12 to 13 nts Requires four different enzymes 1. 2. 3. 4. Exinuclease DNA helicase DNA polymerase DNA ligase Nucleotide excision repair in E. coli Enzyme Protein Function UvrA (MW= 104,000) scans DNA, binds to UvrB UvrB (MW = 78,000) scanner; binds DNA cleaves phosphate bond at 3' end, 5 positions downstream of lesion UvrC (MW = 68,000) binds UvrB & DNA cleaves phosphate bond at 5' end, 8 positions upstream of lesion Exinuclease DNA helicase DNA polymerase DNA ligase UvrD DNA polymerase I (= PolA ) Lig removes DNA fragment fills emerging gap seal nick Nucleotide excision repair in E. coli Mechanism • The (UvrA)2:UvrB complex scans DNA • UvrA dimer dissociates from pryimidine dimer. UvrB binds DNA and cuts at 3’ end. • UvrC associates with UvrB and cuts DNA at 5’ end of the P pyrimidine dimer • UvrD DNA helicase removes the DNA fragment • DNA polymerase I fills the gap • DNA ligase seals the remaining nick. UvrA UvrA UvrB exinuclease P ATP P P OH DNA pol. I UvrD DNA helicase P DNA ligase • Cause: Xeroderma pigmentosum – Defect in human nucleotide excision repair (NER) – 16 polypeptides involved in NER – NER is the only pathway to remove pyrimidine dimers in humans • Symptoms: – Very light sensitive – High risk of sun-light induced skin cancer – Neurological abnormalities (high rate of oxidative metabolism in neurons) • Observation: Direct repair – UV-damaged bacteria that were subsequently incubated in daylight recovered better than those kept in the dark • Photoreactivation – – – – requires DNA photolyases requires visible light at 300 – 500 nm aka “light repair” Contrast: dark repair (BER, NER, mismatch repair) • Structure – – – – DNA photolyases MW = ~ 54,000 (in E. coli) Generally contain 2 chromophores Chromophore No. 1: always FADH Chromophore No. 2: folate (in E. coli and yeast) • N5,N10-methenyltetrahydrofolylpolyglutamate • Function – bind to pyrimidine dimers – resolve pyrimidine dimers into original bases 1. Mechanism outline Absorption of a photon by MTHFpolyGlu 2. Transfer of excitation energy to FADH – 3. Excited FADH – transfers elecctron to the pyrimidine dimer (unstable dimer radical) 4. Shift of electrons breaks cyclobutane ring 5. Electron is transferred back to the flavin radical to regenerate FADH – 6 O -methylguanine • Mutagenic behavior – pairs with thymine rather than cytosine • Specific Repair Mechanism – – – – – differs from base excision repair requires O6-methylguanine methyltransferase one time reaction (“suicide enzyme”) expensive repair mechanism methylated methyltransferase is a transcriptional activator of its own gene O6-methylguanine DNA methyltransferase active Enz. active Enz. Cys-SH O-CH3 Cys-S-CH3 N N O N N H H H 2N N N R O6-methylguanine nucleotide Source: adapted from Lehninger pg. 957 H 2N N N R guanine nucleotide Transition • Transition vs. transversion Exchange of a purine with a purine or pyrimidine with a pyrimindine base. More common than transversion. Often the result of tautomeric shifts. – GC AT transition • causal agents: e.g. base analog 5’bromouracil – AT GC transition • causal agents: e.g. base analog 2-aminopurine • Transversion Exchange of a purine with a pyrimidine base and vice versa – GC TA or GC CG transversion – AT CG or AT TA transversion 6 O -methylguanine • Mutagenic behavior – pairs with thymine rather than cytosine • Specific Repair Mechanism – – – – – differs from base excision repair requires O6-methylguanine methyltransferase one time reaction (“suicide enzyme”) expensive repair mechanism methylated methyltransferase is a transcriptional activator of its own gene O6-methylguanine DNA methyltransferase active Enz. active Enz. Cys-SH O-CH3 Cys-S-CH3 N N O N N H H H 2N N N R O6-methylguanine nucleotide Source: adapted from Lehninger pg. 957 H 2N N N R guanine nucleotide Inaccurate DNAE. coli repair recA- strain E. coli recA+ strain intensive UV exposure Numerous survivors High mutation rates Few survivors Low mutation rates Error-prone repair • Activated upon: – – • severe DNA damage disruption of DNA replication “SOS-response” – – 1. 2. 3. 4. 5. Inaccurate repair mechanism Requires at least 14 proteins in E. coli Din proteins (damage induced) Rec poteins (recombination) Umu proteins (UV-mutagenesis) Uvr proteins (UV-resistance) Others: SulA, HimA, Ssb, and PolB Genes involved in the SOS response Gene pol B uvr A & uvr B umu C & umu D sul A sul B recA din B ssb uvr D him A rec N din D din F Protein function encoded: DNA polymerase II (polymerization subunit) ABC exinuclease DNA polymerase V inhibits cell division via interaction with FtsZ recA protease, recombination and repair DNA polymerase IV single-stranded binding protein DNA helicase II (DNA -unwinding protein) subunit of integration host factor required for recombinational repair ? ? Damaged DNA during replication • UV-damaged DNA results in collapse of the replication fork during DNA replication • 2 scenarios – Unrepaired lesions Unrepaired DNA breaks 12.4 DNA damage and repair and their role in carcinogenesis • A DNA sequence can be changed by copying errors introduced by DNA polymerase during replication and by environmental agents such as chemical mutagens or radiation • If uncorrected, such changes may interfere with the ability of the cell to function • DNA damage can be repaired by several mechanisms • All carcinogens cause changes in the DNA sequence and thus DNA damage and repair are important aspects in the development of cancer • Prokaryotic and eukaryotic DNA-repair systems are analogous 12.4 General types of DNA damage and causes 12.4 Proofreading by DNA polymerase corrects copying errors Figure 12-20 12.4 Schematic model of the proofreading function of DNA polymerase Figure 12-21 12.4 Chemical carcinogens react with DNA directly or after activation, and the carcinogenic effect of a chemical correlates with its mutagenicity Figure 12-22 12.4 Base deamination leads to the formation of a spontaneous point mutation Figure 12-23 12.4 Mismatch repair of single-base mispairs Figure 12-24 12.4 Chemically modified bases, such as thymine-thymine dimers, are corrected by excision repair Figure 12-25 12.4 Excision repair of DNA by the E. coli UvrABC mechanism Figure 12-26 12.4 End-joining repair of nonhomologous DNA Figure 12-28 12.5 Recombination between homologous DNA sites • Recombination provides a means by which a genome can change to generate new combinations of genes • Homologous recombination allows for the exchange of blocks of genes between homologous chromosomes and thereby is a mechanism for generating genetic diversity • Recombination occurs randomly between two homologous sequences and the frequency of recombination between two sites is proportional to the distance between the sites 12.5 The cross-strand Holliday structure is an intermediate in recombination (part I) Figure 12-29 12.5 The cross-strand Holliday structure is an intermediate in recombination (part II) Figure 12-29