garrett and grisham, biochemistry, third edition chapter 28 dna metabolism: replication,...

Garrett and Grisham, Biochemistry, Third Edition

Chapter 28

DNA Metabolism: Replication, Recombination, and Repair

Biochemistry

by

Reginald Garrett and Charles Grisham


Essential Question

• How is this genetic information in the form of DNA replicated, how is the information rearranged, and how is its integrity maintained in the face of damage?


Outline• How Is DNA Replicated?• What Are the Properties of DNA Polymerases?• How Is DNA Replicated in Eukaryotic Cells?• How Are the Ends of Chromosomes Replicated?• How Are RNA Genomes Replicated?• How Is the Genetic Information Shuffled by Genetic Recombination?• Can DNA Be Repaired?• What Is the Molecular Basis of Mutation?

Special Focus: Gene Rearrangements and Immunology – Is It Possible to Generate Protein Diversity Using Genetic Recombination?


28.1 – How Is DNA Replicated?

• DNA replication is semiconservative

• DNA replication is bidirectional

• Replication requires unwinding of the DNA helix

• DNA replication is semidiscontinuous

• The lagging strand is formed from Okazaki fragments


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.

Figure 28.1Watson and Crick’s famous paper, in its entirety. (Reprinted with permission from Watson,J.D., and Crick,F.H.C., Molecular structure of nucleic acid, 1953. Nature 171:737-738. Copyright 1953 Macmillan Publishers Ltd.)

Figure 28.2Untwisting of DNA strands exposes their bases for hydrogen bonding. Base pairing ensures that appropriate nucleotides are inserted in the correct positions as the new complementary strands are synthesized. By this mechanism, the nucleotide sequence of one strand dictates a complementary sequence in its daughter strand. The original strands untwist by rotating about the axis of the unreplicated DNA double helix.


DNA Replication

The Semiconservative Model • Matthew Meselson and Franklin Stahl showed

that DNA replication results in new DNA duplex molecules in which one strand is from the parent duplex and the other is completely new

• Study Figure 30.4 and understand the density profiles from ultracentrifugation experiments

• Imagine and predict the density profiles that the conservative and dispersive models would show

Figure 28.3Three models of DNA replication prompted by Watson and Crick’s double helix structure of DNA. (a) Conservative: Each strand of the DNA duplex is replicated, and the two newly synthesized strands join to form one DNA double helix while the two parental strands remain associated with each other. The products are completely new DNA duplex and the original DNA duplex. (b) Semiconservative: The two strands separate, and each strand is copied to generate a complementary strand. Each parental strand remins associated with its newly synthesized

complement, so each DNA duplex contains one parental strand and one new strand. (c) Dispersive: This model predicts that each of the four strands in the two daughter DNA duplexes contains both newly synthesized segments and segments derived from the parental strands.

Figure 28.4The Meselson and Stahl experiment demonstrating that DNA replication is semi-conservative. On the left are densitometric traces made of UV absorption photographs taken of the ultracentrifugation cells containing DNA isolated from E. coli grown for various generation time after 15N-labeling. The photographs were taken once the migration of the DNA in the density gradient had reached equilibrium. Density increases from left to right. The peaks reveal the positions of the banded DNA with respect to the density of the solution. The number of generations that the E. coli cells were grown (following 14 generations of 15N density-labeling) is shown down the middle. A schematic representation interpreting the pattern expected of semiconservative replication is shown on the right side of this figure. (Adapted from Meselson,M., and Stahl,F.W., 1958. The replication of DNA. Proeceedings of the National Academy of Sciences, U.S.A. 44:671-682.)


Features of DNA Replication

• DNA replication is bidirectional

– Bidirectional replication involves two replication forks, which move in opposite directions

• DNA replication is semidiscontinuous

– The leading strand copies continuously

– The lagging strand copies in segments (Okazaki fragments) which must be joined

Figure 28.5Bidirectional replication of the E. coli chromosome. (a) If replication is bidirectional, auto-radiograms of radioactively labeled replicating chromosomes should show two replication forks heavily labeled with radioactive thymidine. (b) An autoradiogram of the chromosome from a dividing E. coli cell shows bidirectional replication. (Photo courtesy of David M. Prescott, University of Colorado.)


The Enzymologyof DNA Replication

• If Watson and Crick were right, then there should be 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 needs all four deoxynucleotides, a template and a primer - a ss-DNA (with a free 3'-OH) that pairs with the template to form a short double-stranded region


28.2 – What Are the Properties of DNA Polymerases?

• E. coli cells have several DNA polymerases• E. coli DNA polymerase I has three active sites• E. coli DNA polymerase I is its own proofreader

and editor• E. coli DNA polymerase III holoenzyme

replicates the E. coli chromosome• A DNA polymerase III holoenzyme sits at each

replication fork


DNA Polymerase IReplication occurs 5' to 3'

• Nucleotides are added at the 3'-end of the strand • Pol I catalyzes about 20 cycles of polymerization

before the new strand dissociates from template • 20 cycles constitutes moderate "processivity" • Pol I from E. coli is 928 aa (109 kD) monomer • In addition to 5'-3' polymerase, it also has 3'-5'

exonuclease and 5'-3' exonuclease activities

Figure 28.6 The semidiscontinuous model for DNA replication. Newly synthesized DNA is shown as red. Because DNA polymerases only polymerize nucleotides 5 3, both strands must be synthesized in the 5 3 direction. Thus, the copy of the parental 3 5 strand is synthesized continuously; this newly made strand is designated the leading strand. (a) As the helix unwinds, the other parental strand ( the 5 3, strand) is copied in a discontinuous fashion through synthesis of a series of fragments 1000 to 2000 nucleotides in length, called the Okazaki fragments;

the strand constructed from the Okazaki fragments is called the lagging strands. (b) Because both strands are synthesized in concert by a dimeric DNA polymerase situated at the replication fork, the 5 3parental strand must warp around in trombone fashion so that the unit of the dimeric DNA polymerase replicating it can move along it in the 3 5 direction. This parental strand is copied in a discontinuous fashion because the DNA polymerase must occasionally dissociate from this strand and rejoin it further along. The Okazaki fragments are then covalently joined by DNA ligase to form an uninterrupted DNA strand.


More on Pol I

Why the exonuclease activity? • The 3'-5' exonuclease activity serves a

proofreading function! It removes incorrectly matched bases, so that the polymerase can try again

• See Figures 30.9 and 30.10! Notice how the newly-formed strand oscillates between the polymerase and 3'-exonuclease sites,adding a base and then checking it

Figure 28.7The chain elongation reaction catalyzed by DNA polymerase. DNA polymerase I joins deoxynucleoside monophosphate units to the 3´-OH end of the primer, employing dNTPs as substrates. The 3´-OH carries out a nucleophilic attack on the -phosphoryl group of the incoming dNTP to form a phosphoester bond, and PPi is released. The subsequent hydrolysis of PPi by pyrophosphatase renders the reaction effectively irreversible.

Figure 28.8The 3´5´ exonuclease activity of DNA polymerase I removes nucleotides from the 3’-end of the growing DNA chain.


Even More on Pol INicks and Klenows....

• 5'-exonuclease activity, working together with the polymerase, accomplishes "nick translation"

• Hans Klenow used either subtilisin or trypsin to cleave between residues 323 and 324, separating 5'-exonuclease (on 1-323) and the other two activities (on 324-928, the so-called "Klenow fragment”)

• Tom Steitz has determined the structure of the Klenow fragment - see Figure 30.9

Figure 28.9(a) Ribbon diagram of the -subunit dimer of the DNA polymerase III holoenzyme on B-DNA., viewed down the axis of the DNA. One monomer of the -subunit dimer is colored red and the other yellow. The centrally located DNA is mostly blue. (b) Space-filling model of the-subunit dimer of the DNA polymerase III holoenzyme on B-DNA. One monomer is shown in red, the other in yellow. The B-DNA has one strand colored white and the other blue. The hole formed by the -subunits (diameter 3.5 nm) is large enough to easily accommodate DNA (diameter 2.5nm) with no steric repulsion. The rest of polymerase III holoenzyme (“core” polymerase + -complex) associates with this sliding clamp to form the replicative polymerase (not shown). (Adapted from Kong,X-P., et al, 1992.Three-dimensional structure of the beta subunit of E.coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69:425-437; photos courtesy of John Kuriyan of the Rockefeller University)


Features of ReplicationMostly in E. coli, but many features are general

• Replication is bidirectional • The double helix must be unwound - by helicases • Supercoiling must be compensated - by DNA

gyrase • Replication is semidiscontinuous • Leading strand is formed continuously • Lagging strand is formed from Okazaki fragments -

discovered by Tuneko and Reiji "O"


More Features of Replication

• Read page 994 on chemistry of DNA synthesis

• DNA Pol III uses an RNA primer • A special primase forms the required primer • DNA Pol I excises the primer • DNA ligase seals the "nicks" between

Okazaki fragments (See Figure 30.14 for mechanism)

• See Figure 30.15 for a view of replication fork

Figure 28.10General features of a replication fork. The DNA duplex is unwound by the action of DNA gyrase and helicase, and the single strands are coated with SSB (ssDNA-binding protein). 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 -subunit sliding clamp. DNA polymerase I and DNA ligase act downstream on the lagging strand to remove RNA primers, replace them with DNA, and ligate the Okazaki fragments.


DNA Polymerase III

The "real" polymerase in E. coli • At least 10 different subunits • "Core" enzyme has three subunits - , , and • Alpha subunit is polymerase • Epsilon subunit is 3'-exonuclease • Theta subunit is involved in holoenzyme assembly• The beta subunit dimer forms a ring around DNA • Enormous processivity - 5 million bases!


28.3 – How Is DNA Replicated in Eukaryotic Cells?

• The cell cycle controls the timing of DNA replication

• Eukaryotic cells contain a number of different DNA polymerases

• DNA polymerase d is the principal DNA replicase

Figure 28.11A replication factory “fixed” to a cellular substructure extrudes loops of newly synthesized DNA as parental DNA duplex is fed in from the sides. Parental DNA strands are blue; newly synthesized strands are green; small circles indicate origins of replication. (Adapted from Cook,P.R., 1999. The organization of replication and transcription. Science 284:1790-1795.)

Figure 28.12The eukaryotic cell cycle. The stages of mitosis and cell division define the M phase (M for mitosis). G1 (G for gap, not growth) is typically the longest part of the cell cycle; G1, is characterized by rapid growth and metabolic activity. Cells that are quiescent, that is, not growing and dividing (such as neurons), are said to be in G0. The S phase is the time of DNA synthesis. S is followed by G2, a relatively short period of growth when the cell prepares for cell division. Cell cycle times vary from less than 24 hours (rapidly dividing cell such as the epithelial cells lining the mouth and gut) to hundreds of days.


28.4 – How Are the Ends of Chromosomes Replicated?

• Telomeres, the structures at the ends of eukaryotic chromosomes, consist of 5-8 bp tandemly repeated G-rich nucleotide sequences

• Telomeres are 1-12 kbp long• Telomeres are replicated by an RNA-

dependent DNA polymerase called telomerase


Eukaryotic DNA Replication

Like E. coli, but more complex • Human cell: 6 billion base pairs of DNA to copy • Multiple origins of replication: 1 per 3- 300 kbp • Several known animal DNA polymerases - see

Table 30.4 • DNA polymerase alpha - four subunits,

polymerase (processivity = 200) but no 3'-exonuclease

• DNA polymerase beta - similar to alpha

Figure 28.13Model for initiation of the DNA replication cycle in eukaryotes. ORC is present at the replicators throughout the cell cycle. The pre-replication complex (pre-RC) is assembled through the sequential addition of Cdc6p and MCM proteins during a window of opportunity defined by the state of the cyclin-CDKs. After initiation, a post-RC state is established. (Adapted from Figure 2 in Stillman,B., 1996. Cell cycle control of DNA replication. Science 274:1659-1663.)


Mechanism of Replication

in E. coli • The replisome consists of: DNA-unwinding

proteins, the priming complex (primosome) and two equivalents of DNA polymerase III holoenzyme

• Initiation: DnaA protein binds to repeats in ori, initiating strand separation and DnaB, a helicase delivered by DnaC, further unwinds the DNA. Primase then binds and constructs the RNA primer

Figure 28.14Structure of the PCNA homotrimer. Note that the trimeric PCNA ring of eukaryotes is remarkably similar to its prokaryotic counterpart, the dimeric 2-sliding clamp (Figure 28.9). (a) Ribbon representation of the PCNA trimer with an axial view of a B-form DNA duplex in its center. The molecular mass of each PCNA monomer is 37 kD. (b) Molecular surface of the PCNA trimer with each monomer colored differently. The red spiral represents the sugar-phosphate backbone of a strand of B-form DNA. (Adapted from Figure 3 in Krishna,T.S., et at., 1994. Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79:1233-1243. Photos courtesy of John Kuriyan of the Rockefeller University.)


Replication Mechanism IIElongation and Termination

• Elongation involves DnaB helicase unwinding, SSB binding to keep strands separated, and DNA polymerase replicating each strand

• Termination: the "ter" locus, rich in Gs and Ts, signals the end of replication. A Ter protein is also involved. Ter protein is a contrahelicase and prevents unwinding

• Topoisomerase II (DNA gyrase) relieves supercoiling that remains

Figure 28.15Telomere replication. (a) In replication of the lagging strand, short RNA primers are added (pink) and extended by DNA polymerase. When the RNA prirmer at the 5-end of each strand is removed, there is no nucleotide sequence to read in the next round of DNA replication. The result is a gap (primer gap) at the 5-end of each strand (only one end of the chromosome is shown in this figure). (b) Asterisks indicate sequences at the 3-end that cannot be copied by conventional DNA replication. Synthesis of telomeric DNA by telomerase extends the 5-ends of DNA strands, allowing the strands to be copied by normal DNA replication.


28.5 – 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 serves as a template for DNA synthesis

• The RNA-directed DNA polymerase is called Reverse Transcriptase

Figure 28.16The structures of AZT (3-azido-2,3-dideoxythymidine). This nucleoside was the first approved drug for treatment of AIDS. AZT is phosphorylated in vivo to give AZTTP (AZT 5-triphosphate), a substrate analog that binds to HIV reverse transcriptase, HIV reverse transcriptase incorporates AZTTP into growing DNA chains in place of dTTP. Incorporated AZTMP blocks further chain elongation because its 3-azido group cannot form a phosphodiester bond with an incoming nucleotide. Host cell DNA polymerases have little affinity for AZTTP.


28.6 – 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

• Homologous recombination is achieved by the process of general recombination

• General recombination requires the breakage and reunion of DNA strands

• The proteins responsible include RecA, RecBCD, RuvA, RuvB, & RuvC


Another Way to Make DNA

RNA-Directed DNA Polymerase • 1964: Howard Temin notices that DNA

synthesis inhibitors prevent infection of cells in culture by RNA tumor viruses. Temin predicts that DNA is an intermediate in RNA tumor virus replication

• 1970: Temin and David Baltimore (separately) discover the RNA-directed DNA polymerase - aka "reverse trascriptase"


Reverse Transcriptase

• Primer required, but a strange one - a tRNA molecule that the virus captures from the host

• RT transcribes the RNA template into a complementary DNA (cDNA) to form a DNA:RNA hybrid

• All RNA tumor viruses contain a reverse transcriptase


Reverse Transcriptase Activities • Three enzyme activities

– RNA-directed DNA polymerase

– RNase H activity - degrades RNA in the DNA:RNA hybrids

– DNA-directed DNA polymerase - which makes a DNA duplex after RNase H activity destroys the viral genome

• HIV therapy: AZT (or 3'-azido-2',3'- dideoxythymidine) specifically inhibits RT


More Eukaryotic polymerases

• DNA polymerase gamma - DNA-replicating enzyme of mitochondria

• DNA polymerase delta has a 3'-exonuclease as well as proliferating cell nuclear antigen (PCNA)

• PCNA give delta unlimited processivity and is homologous with prokaryotic pol III

• DNA polymerase epsilon - highly processive, but does not have a subunit like PCNA

Figure 28.17Meselson and Weigle’s experiment demonstrated that a physical exchange of chromosome parts actually occurs during recombination. Density-labeled, “heavy” phage, symbolized as ABC phage in the diagram, were used to coinfect bacteria along with ”light” phage, the abc phage. The progeny from the infection were collected and subjected to CsCl density gradient centrifugation. Parental-type ABC and abc phage were well separated in the gradient, but recombinant phage (ABc,Abc, aBc,aBC, and so on ) were distributed diffusely between the two parental bands because they contained chromosomes constituted from fragments of both “heavy” and “light” DNA. These recombinant chromosomes formed by breakage and reunion of parental “heavy” and “light” chromosomes.

Figure 28.18The generation of progeny bacteriophage of two different genotypes from a single phage particle carrying a heteroduplex DNA region within its chromosome. The heteroduplex DNA is composed of one strand that is genotypically XYZ (the + strand), and the other strand that is genotypically XyZ (the - strand). That is, the genotype of the two parental strands for gene Y is different (one is Y, the other y).

Figure 28.19The Holliday model for homologous recombination. The + signs and – signs label strands of like polarity. For example, assume that the two strands running 5´3´ as read left to right are labeled +; and the two strands running 3´5´ as read left to right are labeled-. Only strands of like polarity exchange DNA during recombination. (See text for detailed description.)

Figure 28.20Model of RecBCD-dependent initiation of recombination. (a) RecBCD binds to a duplex DNA end, and its helicase activity begins to unwind the DNA double helix. “Rabbit ears” of ssDNA loop out from RecBCD because the rate of DNA unwinding exceeds the rate of ssDNA release by RecBCD. (b) As it unwinds the DNA, SSB ( and some RecA) bind to the single-stranded regions; the RecBCD endonuclease activity randomly cleaves the ssDNA, showing a greater tendency to cut the 3-terminal strand rather that the 5-terminal strand. (c) When RecBCD encounters a properly oriented site, the 3-terminal strand is cleaved just below the 3-end of . (d) RecBCD now directs the binding of RecA to the 3-terminal strand, as RecBCD endonuclease activity now acts more often on the 5-terminal strand. (e) A nucleoprotein filament consisting of RecA-coated 3’-strand ssDNA is formed. This nucleoprotein filament is capable of homologous pairing with a dsDNA and strand invasion. (Adapted from Figure 2 in Eggleston,A.D., and West,S.C., 1996. Exchanging partners: recombination in E.coli. Trends in Genetics 12:20-25; and Figure 3 in Eggleson,A.K. and West,S.C., 1997. Recombination initiation: Easy as A,B,C,D ….? Current Biology 7:R745--R749.)

Figure 28.21The structure of RecA, a 352-residue, 38-kD protein. (a) Ribbon diagram of the RecA monomer. Note the ADP bound at the site near helices C and D. (b) RecA filament. Four turns of a helical filament that has six RecA monomers per turn. A RecA monomer is highlighted in red. (Adapted from Figures 2 and 3 in Roca, A.I., and Cox,M.M., 1997. RecA protein: Structure, function, and role in recombinational DNA repair. Progress in Nucleic Acid Research and Molecular Biology 56:127-223. Photos courtesy of Michael M. Cox, University of Wisconsin.)

Figure 28.22Model for homologous recombination as promoted by RecA enzyme. (a) RecA protein (and SSB) aid strand invasion of the 3’-ssDNA into a homologous DNA duplex, (b) forming a D-loop. (c) The D-loop strand that has been displaced by strand invasion pairs with its complementary strand in the original duplex to form a Holiday junction as strand invasion continues.

Figure 28.24The typical transposon has inverted nucleotide-sequence repeats at its termini, represented here as the 12-bp sequence ACGTACGTACGT (a). It acts at a target sequence (shown here as the sequence CATGC) within host DNA by creating a staggered cut (b) whose protruding single-stranded ends are then ligated to the transposon (c). The gaps at the target site are then filled in, and the filled-in strands are ligated (d). Transposon insertion thus generates direct repeats of the target site in the host DNA, and these direct repeats flank the inserted transposon.


28.7 – Can DNA Be Repaired?

A fundamental difference from RNA, protein, lipid, etc.

• All these others can be replaced, but DNA must be preserved

• Cells require a means for repair of missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links

• Two principal mechanisms: mismatch repair and methods for reversing chemical damage

Figure 28.23


Mismatch Repair

• Mismatch repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it

• Methyl-directed pathway of E. coli is an example

• Since methylation occurs post-replication, repair proteins identify methylated strand as parent, remove mismatched bases on other strand and replace them

Figure 28.25UVA 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.


Reversing Chemical Damage

• Pyrimidine dimers can be repaired by photolyase

• Excision repair: DNA glycosylase removes damaged base, creating an "AP site"

• AP endonuclease cleaves backbone, exonuclease removes several residues and gap is repaired by DNA polymerase and DNA ligase

Figure 28.26Base excision repair. A damaged base (■) is excised from the sugar-phosphate backbone by DNA glycosylase, creating an AP site. Then, an apurinic/apyrimidinic endonuclease severs the DNA strand, and an excision nuclease removes the AP site and several nucleotides. DNA polymerase I and DNA ligase then repair the gap.


28.8 – What Is the Molecular Basis of Mutation?

• Point mutations arise by inappropriate base-pairing

• Mutations can be caused by base analogs

• Chemical mutagens react with bases in DNA

• Insertions and deletions

Figure 28.27 Point mutations due to base mispairings. (a) An example based on tautomeric properties. The rare imino tautomer of adenine base pairs cytosine rather than thymine. (1) The normal A-T base pair. (2) The A*-C base pair is possible for the adenine tautomer in which a proton has been transferred from the 6-NH2 of adenine to N-1. (3) Pairing of C with the imino tautomer of A (A*) leads to a transition mutation (A-T to G-C) appearing in the next generation. (b) A in the syn conformation pairing with G (G is in the usual anti conformation). (c) T and C form a base pair by H-bonding interactions medicated by a water molecule.

Figure 28.285-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.

Figure 28.29(a) 2-Aminopurine normally base-pairs with T but (b) may also pair with cytosine through a single hydrogen bond.

Figure 28.30Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an A-T to G-C transition.

Figure 28.31Chemical mutagens. (a) HNO2 (nitrous acid) converts cytosine to uracil and adenine to hypoxanthine. (b) Nitrosoamines, organic compounds that react to form nitrous acid, also lead to the oxidative deamination of A and C. (c) Hydroxylamine (NH2OH) reacts with cytosine, converting it to a derivative that base-pairs with adenine instead of guanine. The result is a C-G to T-A transition. (d) Alkylation of G residues to give O6-methylguanine, which base-pairs with T. (e) Alkylating agents include nitrosoamines, nitrosoguanidines, nitrosoureas, alkyl sulfates, and nitrogen mustards. Note that nitrosoamines are mutagenic in two ways: They can react to yield HNO2, or they can act as alkylating agents. The nitrosoguanidine, N-methyl-N-nitro-N-nitrosoguanidine, is a very potent mutagen used in laboratories to induce mutations in experimental organisms such as Drosophila melanogaster. Ethylmethane sulfonate (EMS) and dimethyl sulfate are also favorite mutagens among geneticists.


Gene Rearrangements and Immunology

• Cells active in the immune response are capable of gene rearrangement

• IgG molecules, the major class of circulating antibodies, are encoded by rearranged genes

• DNA rearrangements assemble an L-chain gene from 3 separate genes

• DNA rearrangements assemble an H-chain gene from 4 separate genes

Figure 28.32Diagram of the organization of the IgG molecule. Two identical L chains are joined with two identical H chains. Each L chain is held to an H chain via an interchain disulfide bond. The variable regions of the four polypeptides lie at the ends of the arms of the Y-shaped molecule. These regions are responsible for the antigen recognition function of the antibody molecules. The actual antigen-binding site is constituted from hypervariable residue within the VL and VH regions. For purposes of illustration, some features are shown on only one of the other L chain or H chain, but all features are common to both chains.

Figure 28.33The characteristic “collapsed -barrel domain” known as the immunoglobulin fold. The -barrel structures for both (a) variable and (b) constant regions are shown. (c) A schematic diagram of the 12 collapsed -barrel domains that make up an IgG molecule. CHO indicates the carbohydrate addition site; Fab denotes one of the two antigen-binding fragments of IgG, and Fc, the proteolytic fragment consisting of the pairs of CH2 and CH3 domains.

Figure 28.34Organization of human immunoglobulin gene segments. Green, orange, blue or purple colors indicate the exons of a particular VL or VH gene. (a) L-chain gene assembly: During B-lymphocyte maturation in the bone marrow, one of the 40 V genes combines with one of the 5J genes and is joined with a C gene. During the recombination process, the intervening DNA between the gene segments is deleted (see Figure 28.36). These rearrangements occur by a mostly random process, giving rise to many possible light-chain sequences from each gene family. (b) H-chain gene assembly: H chains are encoded by V,D,J. and C genes. In H-chain gene rearrangements, a D gene joins with a J gene and then one of the V genes adds to the DJ assembly. (Adapted from Figure 2b and c in Nossal, G.J.V., 2003. The double helix and immunology. Nature 421:440-444.)

Figure 28.35Consensus elements are located above and below germline variable-region genes that recombine to form genes encoding immunoglobulin chains. These consensus elements are complementary and are arranged in a heptamer-nonamer,12-to 23-bp spacer pattern. (Adapted from Tonewaga,S.,1983. Somatic generation of antibody diversity. Nature 302:575.)

Figure 28.36 Model for V(D)J recombination. A RAG1: RAG2 complex is assembled on DNA in the region of recombination signal sequences (a), and this complex introduces double-stranded breaks in the DNA at the borders of protein-coding sequences and the recombination signal sequences (b). The products of RAG1:RAG2 DNA cleavage are novel: The DNA bearing the recombination signal sequences has blunt ends, whereas the coding DNA has hairpin ends. That is, the two strands of the V and J coding DNA segments are covalently joined as result of transesterification reactions catalyzed by RAG1:RAG2. To complete the recombination process, the two RSS ends are precisely joined to make a covalently closed circular dsDNA, but the V and J coding ends undergo further processing before they are joined (c). Coding-end processing involves opening of the V and J hairpins and the addition or removal of nucleotides from the

strands. This processing means that joining of the V and J coding ends is imprecise, providing an additional means for introducing antibody diversity. Finally, the V and J coding segments are then joined to create a recombinant immunoglobulin-encoding gene (d). The processing and joining reactions require RAG1:RAG2, DNa-dependent protein kinase (DNA-PK, which consists of three subunits-Ku70,Ku80, and DNA-PKCS), and DNA ligase. (Adapted

from Figure 1 in Weaver,D.T., and Alt,F.W., 1997. From RAGs to stitches. Nature 388:428-429.)

Figure 28.37Recombination between the VK and JK genes can vary by several nucleotides, giving rise to variations in amino acid sequence and hence diversity in immunoglobulin L chains.

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