Biochemistry, 4th Edition P94 ppsx

The protein splicing function of inteins is found in its N-terminal and its C-terminal regions; the endonuclease function that carries out par-asitic insertion of the intein sequence int

(Figure 28.30) Less often, 5-BU is inserted into DNA at cytosine sites, not T sites.

Then, if it base-pairs in its keto form, mimicking T, a C–G to T–A transition ensues

The adenine analog, 2-aminopurine (recall that adenine is 6-aminopurine) normally

behaves like A and base-pairs with T However, 2-AP can form a single H bond of

suf-ficient stability with cytosine (Figure 28.31) that occasionally C replaces T in DNA

replicating in the presence of 2-AP Hypoxanthine (Figure 28.32) is an adenine

ana-log that arises in situ in DNA through oxidative deamination of A Hypoxanthine

base-pairs with cytosine, creating an A–T to G–C transition

Chemical Mutagens React with the Bases in DNA

Chemical mutagens are agents that chemically modify bases so that their

base-pairing characteristics are altered For instance, nitrous acid (HNO2) causes the

ox-idative deamination of primary amine groups in adenine and cytosine Oxox-idative

deamination of cytosine yields uracil, which base-pairs the way T does and gives a

C–G to T–A transition (Figure 28.33a) Hydroxylamine specifically causes C–G to

T–A transitions because it reacts specifically with cytosine, converting it to a

deriva-tive that base-pairs with adenine instead of guanine (Figure 28.33c) Alkylating

agents(Figure 28.33e) are also chemical mutagens Alkylation of reactive sites on the

bases to add methyl or ethyl groups alters their H bonding and hence base pairing

For example, methylation of O6on guanine (giving O6-methylguanine) causes this

G to mispair with thymine, resulting in a G–C to A–T transition (Figure 28.33d)

Alkylating agents can also induce point mutations of the transversion type Alkylation

of N7 of guanine labilizes its N-glycosidic bond, which leads to elimination of the

purine ring, creating a gap in the base sequence An enzyme, AP endonuclease, then

cleaves the sugar–phosphate backbone of the DNA on the 5-side, and the gap can

be repaired by enzymatic removal of the 5-sugar–phosphate and insertion of a new

nucleotide (see Figure 28.28) A transversion results if a pyrimidine nucleotide is

in-serted in place of the purine during enzymatic repair of this gap

Insertions and Deletions

The addition or removal of one or more base pairs leads to insertion or deletion

mu-tations, respectively Either shifts the triplet reading frame of codons, causing

frameshift mutations(misincorporation of all subsequent amino acids) in the protein

encoded by the gene Such mutations can arise if flat aromatic molecules such as

acri-dine orange insert themselves between successive bases in one or both strands of the

double helix This insertion or, more aptly, intercalation, doubles the distance

be-tween the bases as measured along the helix axis (see Figure 11.12) This distortion

of the DNA results in inappropriate insertion or deletion of bases when the DNA is

replicated Disruptions that arise from the insertion of a transposon within a gene also

fall in this category of mutation (see Figure 28.23)

Prions Are Proteins That Can Act as Genetic Agents

DNA is the genetic material in organisms, although some viruses have RNA genomes

The idea that proteins could carry genetic information was considered early in the

history of molecular biology and dismissed for lack of evidence Prions may be an

ex-ception to this rule

Prionis an acronym derived from the words proteinaceous infectious particle The

term prion was coined to distinguish such particles, which are pathogenic and thus

capable of causing disease, from nucleic acid–containing infectious particles such

as viruses and virions Prions are transmissible agents (genetic material?) that are

apparently composed only of a protein that has adopted an abnormal

conforma-tion They produce fatal degenerative diseases of the central nervous system in

N N

H

H N

O

N

N N

O C1

C  1

2-Aminopurine (2-AP) Thymine (b)

H

1 6 2

5 4 9

8 7

H

N N

H N

N

N

N N

O C1

C  1

H

H N

N

FIGURE 28.31 (a) 2-Aminopurine normally base-pairs with T but (b) may also pair with cytosine through a

single hydrogen bond.

Cytosine

H

O

N H N O H

N N N

C  1

C  1

N H

1 6

N N

Oxidative deamination HN N

N N O

Hypoxanthine

(Hypoxanthine is in its keto tautomeric form here) N

NH2

FIGURE 28.32 Oxidative deamination of adenine in DNA yields hypoxanthine, which base-pairs with cytosine, resulting in an A–T to G–C transition.

C1

C1

H N

N N N N H

O

H

Pairs

normally

with

cytosine

Guanine

Alkylating agent

H N

N N N

N O

H

Sometimes

pairs

with

thymine

O6 -methylguanine

Nitrosoamines:

Dimethylnitrosoamine

N

H3C

Diethylnitrosoamine

H3C

N

CH3

CH3

CH2

CH2

Nitrosoguanidine:

CH3

C NH N H

NO2

N-methyl-N

-nitro-N-nitrosoguanidine

Nitrosourea:

Ethyl nitrosourea

O C

CH2CH3

Nitrogen Mustard:

N

H3C

CH2 CH2 Cl

NH2 CH2 Cl

Alkyl Sulfates:

Ethylmethane sulfonate

O

H3C CH2CH3 O

Dimethyl sulfate

O

H3C

O

CH3 O

(a)

HNO2 H

N

N

H N

O

C1

C  1

H

N N O

N N

N H

(c)

C  1

N N

H

H

N

N

N

O C 1

C  1

H N

H

HNO2

N

N N

C  1

N

Cytosine

NH2OH H

N N

H N

O

C1

C  1

H

N N O

N N

N H

C  1

O

H

N

(b) Generic structure of nitrosoamines

N N

R1

R2

FIGURE 28.33 Chemical 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 (NH2 OH) reacts with cytosine,

convert-ing 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,

nitro-soguanidines, nitrosoureas, alkyl sulfates, and nitrogen

mustards Note that nitrosoamines are mutagenic in two

ways: They can react to yield HNO 2 , or they can act as

alkylating agents The nitrosoguanidine, N-methyl-N

-nitro-N-nitrosoguanidine, is a very potent mutagen used in

labora-tories to induce mutations in experimental organisms such

as Drosophila melanogaster Ethylmethane sulfonate (EMS)

and dimethyl sulfate are also favorite mutagens among

geneticists.

SPECIAL FOCUS

mammals and are believed to be the agents responsible for the human diseases

kuru, Creutzfeldt-Jakob disease, Gerstmann-Straussler-Sheinker syndrome, and fatal

familial insomnia Prions also cause diseases in animals, including scrapie (in

sheep), “mad cow disease” (bovine spongiform encephalopathy), and chronic

wast-ing disease (in elk and mule deer) All attempts to show that the infectivity of these

diseases is due to a nucleic acid–carrying agent have been unsuccessful Prion

dis-eases are novel in that they are genetic and infectious; their occurrence may be

spo-radic, dominantly inherited, or acquired by infection

PrP, the prion protein, comes in various forms, such as PrP c ,the normal cellular

prion protein, and PrP sc ,the scrapie form of PrP, a conformational variant of PrPc

that is protease resistant, sometimes written as PrPres These two forms are thought

to differ only in terms of their secondary and tertiary structure One model suggests

that PrPcis dominated by -helical elements (Figure 28.34a), whereas PrPschas both

-helices and -strands (Figure 28.34b) It has been hypothesized that the presence

of PrPsccan cause PrPcto adopt the PrPscconformation The various diseases are a

consequence of the accumulation of the abnormal PrPscform, which accumulates as

amyloid plaques (amyloid starchlike), that cause destruction of tissues in the

cen-tral nervous system Ironically, recent evidence suggests that PrPcmay function as a

nucleic acid-binding protein The 1997 Nobel Prize in Physiology or Medicine was

awarded to Stanley B Prusiner for his discovery of prions

ANIMATED FIGURE 28.34 Speculative

models suggest that (a) PrPc is mostly -helical, whereas

(b) PrPsc has both -helices and -strands.(Adapted from Figure 1 in Prusiner, S B., 1996 Molecular biology and the

patho-genesis of prion diseases Trends in Biochemical Sciences

21:482–487.) See this figure animated at www.cengage com/login.

Gene Rearrangements and Immunology—Is It Possible to Generate

Protein Diversity Using Genetic Recombination?

Animals have evolved a way to exploit genetic recombination in order to generate

protein diversity This development was crucial to the evolution of the immune

sys-tem For example, the immunoglobulin genes are a highly evolved system for

max-imizing protein diversity from a finite amount of genetic information This diversity

A DEEPER LOOK

Inteins—Bizarre Parasitic Genetic Elements Encoding a Protein-Splicing Activity

Inteins are parasitic genetic elements found within

protein-coding regions of genes These selfish DNA

el-ements are transcribed and translated along with the

flanking host gene sequences The typical intein

pro-tein consists of two domains: One domain is capable of

self-catalyzed protein splicing; the other is an

endo-nuclease that mediates the insertion of the intein

nu-cleotide sequence into host genes After the full protein

is synthesized, the intein catalyzes excision of itself from

the host protein and ligation of adjacent host

polypep-tide regions to form the functional protein that the

host gene encodes These adjacent polypeptides are

termed exteins (“external proteins”) to distinguish

them from the intein (“internal protein”) Inteins have

been found across all domains of life—archaea,

bacte-ria, and eukaryotes—although thus far only in

unicel-lular organisms Inteins vary in size from about 130 to

600 amino acid residues The protein splicing function

of inteins is found in its N-terminal and its C-terminal

regions; the endonuclease function that carries out

par-asitic insertion of the intein sequence into host genes is

found in the central part of the intein Splicing of the

protein is an intramolecular process that liberates the

intein sequence and ligates the host protein sequences

(see accompanying figure)

Inteins are usually found as inserts in highly conserved

host genes that have essential functions, such as genes

en-coding DNA or RNA polymerases, proton-translocating

ATPases, or other vital metabolic enzymes Their location

in such genes means that removal of the intein via

dele-tion or genetic rearrangement is more difficult The

endo-nuclease activity of the intein recognizes a 14– to

40–base-pair sequence in a potential host gene and cleaves the

DNA there During repair of the double-stranded DNA

break, the intein gene is copied into the cleavage site,

thereby establishing the parasitic genetic element in the

host gene

C-extein Translation

Transcription

N-extein

Intein

N-extein

N-extein

Protein

Protein Splicing:

mRNA DNA

Intein

Coding sequence Coding sequence Intein

Host gene and intein

N

N

NH2 H H

O

O O

Intein

C-extein N

H

NH2

H2N O

H2N OH

OH

O C

C O

C-extein N

H

NH2

O O

O O

1

2

3

4

O

H2N

NH

H2N

O NH



N-extein

C

N-extein

C

C-extein O

C-extein OH

O

H2N

N H

Intact Host Protein

O

Excised Intein

O

䊳 Transcription and translation of the combined intein-host

gene flanking sequences leads to synthesis of a fused intein–

extein protein The intein splices itself out when (1) the

C-terminal residue of the N-extein is shifted to the O (or S)

atom of a neighboring intein Ser (or Cys) residue, (2) the

N- extein C-terminal carbonyl undergoes nucleophilic attack

by the O (or S) atom of a Ser (or Cys) residue at the end of

the C-extein in a transesterification reaction that creates a

branched protein intermediate, (3) cyclization of the intein

C-terminal asparagine residue excises the intein, and (4) the

two exteins are properly united via a peptide bond when the

N-extein C-terminus spontaneously shifts to the C-extein

N-terminus to form an intact host protein.

Adapted from Paulus, H., 2000 Protein splicing and related

forms of protein autoprocessing Annual Review of Biochemistry

69:447–496; and Gogarten, J P., et al., 2002 Inteins: Structure,

function, and evolution Annual Review of Microbiology 56:263–287.

is essential for gaining immunity to the great variety of infectious organisms and

for-eign substances that cause disease

Cells Active in the Immune Response Are Capable of Gene

Rearrangement

Only vertebrates show an immune response If a foreign substance, called an

antigen,gains entry to the bloodstream of a vertebrate, the animal responds via a

protective system called the immune response The immune response involves

pro-duction of proteins capable of recognizing and destroying the antigen This

re-sponse is mounted by certain white blood cells—the B- and T-cell lymphocytes and

the macrophages B cells are so named because they mature in the bone marrow;

T cells mature in the thymus gland Each of these cell types is capable of gene

arrangement as a mechanism for producing proteins essential to the immune

re-sponse Antibodies, which can recognize and bind antigens, are immunoglobulin

proteins secreted from B cells Because antigens can be almost anything, the

im-mune response must have an incredible repertoire of structural recognition Thus,

vertebrates must have the potential to produce immunoglobulins of great diversity

in order to recognize virtually any antigen

Immunoglobulin G Molecules Contain Regions of Variable

Amino Acid Sequence

Immunoglobulin G (IgG or␥-globulin) is the major class of antibody molecules

found circulating in the bloodstream IgG is a very abundant protein, amounting to

12 mg per mL of serum It is a 150-kD 22-type tetramer The  or H (for heavy)

chain is 50 kD; the  or L (for light) chain is 25 kD A preparation of IgG from

serum is heterogeneous in terms of the amino acid sequences represented in its L

and H chains However, the IgG L and H chains produced from any given B

lym-phocyte are homogeneous in amino acid sequence L chains consist of 214 amino

acid residues and are organized into two roughly equal segments: the VLand CL

re-gions The VLdesignation reflects the fact that L chains isolated from serum IgG

show variations in amino acid sequence over the first 108 residues, VLsymbolizing

this “variable” region of the L polypeptide The amino acid sequence for residues

109 to 214 of the L polypeptide is constant, as represented by its designation as the

“constant light,” or CL, region The heavy, or H, chains consist of 446 amino acid

residues Like L chains, the amino acid sequence for the first 108 residues of H

polypeptides is variable, ergo its designation as the VHregion, while residues 109 to

446 are constant in amino acid sequence This “constant heavy” region consists of

three quite equivalent domains of homology designated CH1, CH2, and CH3 Each

L chain has two intrachain disulfide bonds: one in the VLregion and the other in

the CLregion The C-terminal amino acid in L chains is cysteine, and it forms an

in-terchain disulfide bond to a neighboring H chain Each H chain has four intrachain

disulfide bonds, one in each of the four regions Figure 28.35 presents a diagram of

IgG organization Within the variable regions of the L and H chains, certain

posi-tions are hypervariable with regard to amino acid composition These hypervariable

residues occur at positions 24 to 34, 50 to 55, and 89 to 96 in the L chains and at

positions 31 to 35, 50 to 65, 81 to 85, and 91 to 102 in the H chains The

hypervari-able regions are also called complementarity-determining regions, or CDRs, because

it is these regions that form the structural site that is complementary to some part

of an antigen’s structure, providing the basis for antibody⬊antigen recognition

In the immunoglobulin genes, the arrangement of exons correlates with protein

structure In terms of its tertiary structure, the IgG molecule is composed of 12

dis-crete collapsed -barrel domains Within each domain, alternating -strands are

anti-parallel to one another, a pattern known by the name Greek key motif The

charac-teristic structure of this domain is referred to as the immunoglobulin fold (Figure

28.36) Each of IgG’s two heavy chains contributes four of these domains and each

S S

S S

S S

S S

S S

S S

S S

S S

N N

S S S S SS S

C (CH2O)n addition site

C

CH1

VH

VL

CL

Hinge region

N

NHeavy Light

214

Antigen binding

Antigen binding

4.5 nm

FIGURE 28.35 Diagram 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

(purple) of the four polypeptides lie at the ends of the

arms of the Y-shaped molecule These regions are

re-sponsible for the antigen recognition function of the

antibody molecules The actual antigen-binding site is

constituted from hypervariable residues within the V L

and V H regions For purposes of illustration, some

fea-tures are shown on only one or the other L chain or H

chain, but all features are common to both chains.

N

Immunoglobulin

VL domain

(a)

9

Immunoglobulin

CL domain

(~ C ~ domains)

(b)

7

6

8

3 4 5

CL

COO–

COO–

CHO CHO

–OOC

–OOC

CL

CH1

CH1

Fab

Fc

Antigen-binding site

Antigen-binding site

(c)

H3N

3

NH3

+

+

+

+

6

3 8 9

ACTIVE FIGURE 28.36

The 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; F ab denotes one

of the two antigen-binding fragments of IgG, and F c , the proteolytic fragment con-sisting of the pairs of C H 2 and C H 3 domains.

Test yourself on the concepts in this fig-ure at www.cengage.com/login.

of its light chains contributes two The four variable-region domains (one on each

chain) are encoded by multiple exons, but the eight constant-region domains are

each the product of a single exon All of these constant-region exons are derived from

a single ancestral exon encoding an immunoglobulin fold The major

variable-region exon probably derives from this ancestral exon also Contemporary

immuno-globulin genes are a consequence of multiple duplications of the ancestral exon

The discovery of variability in amino acid sequence in otherwise identical

poly-peptide chains was surprising and almost heretical to protein chemists For

geneti-cists, it presented a genuine enigma They noted that mammals, which can make

millions of different antibodies, don’t have millions of different antibody genes

How can the mammalian genome encode the diversity seen in L and H chains?

The Immunoglobulin Genes Undergo Gene Rearrangement

The answer to the enigma of immunoglobulin sequence diversity is found in the

or-ganization of the immunoglobulin genes The genetic information for an

immuno-globulin polypeptide chain is scattered among multiple gene segments along a

chro-mosome in germline cells (sperm and eggs) During vertebrate development and the

formation of B lymphocytes, these segments are brought together and assembled by

DNA rearrangement(that is, genetic recombination) into complete genes DNA

re-arrangement, or gene reorganization, provides a mechanism for generating a variety

of protein isoforms from a limited number of genes DNA rearrangement occurs in

only a few genes, namely, those encoding the antigen-binding proteins of the immune

response—the immunoglobulins and the T-cell receptors The gene segments

encod-ing the amino-terminal portion of the immunoglobulin polypeptides are also

unusu-ally susceptible to mutation events The result is a population of B cells whose

antibody-encoding genes collectively show great sequence diversity even though a given cell can

make only a limited set of immunoglobulin chains Hence, at least one cell among the

B-cell population will likely be capable of producing an antibody that will specifically

recognize a particular antigen

DNA Rearrangements Assemble an L-Chain Gene by Combining

Three Separate Genes

The organization of various immunoglobulin gene segments in the human genome

is shown in Figure 28.37 L-chain variable-region genes are assembled from two kinds

of germline genes: V L and J L( J stands for joining) In mammals, there are two

differ-ent families of L-chain genes: the␬, or kappa, gene family and the ␭, or lambda, gene

family;each family has V and J members These families are on different

chromo-somes Humans have 40 functional V genes and 5 functional J genes for the  light

chains and 31 V genes and 4 J genes for the  light chain The V and J genes lie

up-stream from the single C␬genethat encodes the L-chain constant region Each V

gene has its own Lsegment for encoding the L-chain leader peptide that targets the

L chain to the endoplasmic reticulum for IgG assembly and secretion (This leader

peptide is cleaved once the L chain reaches the ER lumen.) The  family of L-chain

genes is organized similarly (Figure 28.37) In different mature B-lymphocyte cells,

V and J genes have joined in different combinations, and along with the C–V

gene, form complete L–V chains with a variety of V regions However, any given

B lymphocyte expresses only one V–J combination Construction of the mature

B-lymphocyte L-chain gene has occurred by DNA rearrangements that combine three

genes (L–V,, J,, C,) to make one polypeptide!

DNA Rearrangements Assemble an H-Chain Gene by Combining

Four Separate Genes

The first 98 amino acids of the 108-residue, H-chain variable region are encoded by

a V H gene. Each VHgene has an accompanying LHgene that encodes its essential

leader peptide It is estimated that there are 200 to 1000 V genes and that they can

be subdivided into eight distinct families based on nucleotide sequence homology The members of a particular VHfamily are grouped together on the chromosome, separated from one another by 10 to 20 bp In assembling a mature H-chain gene,

a VHgene is joined to a D gene (D for diversity), which encodes amino acids 99 to

113 of the H chain These amino acids comprise the core of the third CDR in the variable region of H chains The VH–D gene assemblage is linked in turn to a

J H gene, which encodes the remaining part of the variable region of the H chain The VH, D, and JHgenes are grouped in three separate clusters on the same chro-mosome The four JHgenes lie 7 kb upstream of the eight C genes, the closest of which is C Any of four C genes may encode the constant region of IgG H chains:

C1, C2a, C2b, and C3 Each C gene is composed of multiple exons (only C is shown in Figure 28.37, none of the other C genes) Ten to twenty D genes are found

1 to 80 kb farther upstream The VHgenes lie even farther upstream In B lympho-cytes, the variable region of an H-chain gene is composed of one each of the LH–VH

genes, a D gene, and a JHgene joined head to tail Because the H-chain variable re-gion is encoded in three genes and the joinings can occur in various combinations, the H chains have a greater potential for diversity than the L-chain variable regions that are assembled from just two genes (for example, L–V and J) In making H-chain genes, four genes have been brought together and reorganized by DNA re-arrangement to produce a single polypeptide!

V–J and V–D–J Joining in Light- and Heavy-Chain Gene Assembly

Is Mediated by the RAG Proteins

Specific nucleotide sequences adjacent to the various variable-region genes suggest

a mechanism in which these sequences act as joining signals All germline V and D genes are followed by a consensus CACAGTG heptamer separated from a consen-sus ACAAAAACC nonamer by a short, nonconserved 23-bp spacer Likewise, all germline D and J genes are immediately preceded by a consensus GGTTTTTGT nonamer separated from a consensus CACTGTG heptamer by a short noncon-served 12-bp spacer (Figure 28.38) Note that the consensus elements downstream

of a gene are complementary to those upstream from the gene with which it re-combines Indeed, it is these complementary consensus sequences that serve as

recombination signal sequences (RSSs) and determine the site of recombination between variable-region genes Functionally meaningful recombination happens only where one has a 12-bp spacer and the other has a 23-bp spacer (Figure 28.38)

V1

(a)

V1 V2 V3 J C

Germline locus

V J

rearranged

VN

(b)

V H2

Germline locus

V H2

DJ rearranged

V H2

VDJ rearranged

D H

FIGURE 28.37 Organization of human immunoglobulin

gene segments Green, orange, blue, or purple colors

indi-cate the exons of a particular V L or V Hgene (a) L-chain

gene assembly: During B-lymphocyte maturation in the

bone marrow, one of the 40 V genes combines with one

of the 5 J genes and is joined with a C gene During the

recombination process, the intervening DNA between the

gene segments is deleted (see Figure 28.39).These

rear-rangements 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.)

Lymphoid cell-specific recombination-activating gene proteins 1 and 2 (RAG1 and

RAG2) recognize and bind at these RSSs, presumably through looping out of the

12- and 23-bp spacers and alignment of the homologous heptamer and nonamer

re-gions (Figure 28.39) RAG1 and RAG2 together function as the V(D)J recombinase.

RAG1/RAG2 action cleaves and processes the ends of the V and J DNA, producing

 chain

 chain

H chain

23 12

7 9

FIGURE 28.38 Consensus elements are located above and below germline variable-region genes that recom-bine 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.)

(c)

Discarded

(d)

DNA ligase

Recombined gene

DNA–PK complex

23 bp

12 bp

RAG1/RAG2

Nonamer

Heptamer

DNA–PK complex DNA ligase

V

V V

V

FIGURE 28.39 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

bor-ders 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 a 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 (c) Coding-end processing involves opening of the V and J hairpins and the addition or removal of

nucleotides.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

recombi-nant immunoglobulin-encoding gene (d) The processing and joining reactions require RAG1⬊RAG2,

DNA-dependent protein kinase (DNA-PK)—Ku70, Ku80, and DNA ligase (Adapted from Figure 1 in Weaver, D.T., and Alt, F.W.,

what is effectively a double-stranded break (DSB) Proteins involved in NHEJ-type repair of DSBs (Ku70/80, DNA-PK, and DNA ligase) bind at the DSB and religate the DNA to create a recombinant immunoglobulin gene (Figure 28.39)

Imprecise Joining of Immunoglobulin Genes Creates New Coding Arrangements

Joining of the ends of the immunoglobulin-coding regions during gene reorganiza-tion is somewhat imprecise This imprecision actually leads to even greater antibody di-versity because new coding arrangements result Position 96 in  chains is typically

en-coded by the first triplet in the Jelement Most  chains have one of four amino acids

here, depending on which Jgene was recruited in gene assembly However, occasion-ally only the second and third bases or just the third base of the codon for position 96

is contributed by the Jgene, with the other one or two nucleotides supplied by the V

segment (Figure 28.40) So, the precise point where recombination occurs during gene reorganization can vary over several nucleotides, creating even more diversity

Antibody Diversity Is Due to Immunoglobulin Gene Rearrangements

Taking as an example the mouse with perhaps 300 V genes, 4 J genes, 200 VH

genes, 12 D genes, and 4 JHgenes, the number of possible combinations is given by

300 4  200  12  4 Thus, more than 107different antibody molecules can be created from roughly 500 or so different mouse variable-region genes Including the possibility for V–Jjoinings occurring within codons adds to this diversity, as does the high rate of somatic mutation associated with the variable-region genes (Somatic mutations are mutations that arise in diploid cells and are transmitted to the progeny of these cells within the organism, but not to the offspring of the or-ganism.) Clearly, gene rearrangement is a powerful mechanism for dramatically en-hancing the protein-coding potential of genetic information

V G T T C A T C T T C G A

J A T G G C A A G C T T G

Val

94

Gln

Ser Leu

V G T T C A T C T T C G A

J A T G G C A A G C T T G

Val His

Ser Leu

V G T T C A T C T T C G A

J A T G G C A A G C T T G

Val His

V G T T C A T C T T C G A

J A T G G C A A G C T T G

Val His

FIGURE 28.40 Recombination between the Vand J

genes can vary by several nucleotides, giving rise to

variations in amino acid sequence and hence diversity

in immunoglobulin L chains.

SUMMARY

28.1 How Is DNA Replicated? DNA replication is accomplished

through strand separation and the copying of each strand Strand

sepa-ration is achieved by untwisting the double helix Each separated strand

acts as a template for the synthesis of a new complementary strand

whose nucleotide sequence is fixed by Watson–Crick base-pairing rules.

Base pairing then dictates an accurate replication of the original DNA

double helix DNA replication follows a semiconservative mechanism

where each original strand is copied to yield a complete complementary

strand and these paired strands, one old and one new, remain together

as a duplex DNA molecule Replication begins at specific regions called

origins of replication and proceeds in both directions Bidirectional

replication involves two replication forks, which move in opposite

di-rections Helicases unwind the double helix, and DNA gyrases act to

overcome torsional stress by introducing negative supercoils at the

ex-pense of ATP hydrolysis Because DNA polymerases synthesize DNA

only in a 5→3 direction, replication is semidiscontinuous: The 3→5

strand can be copied continuously by DNA polymerase proceeding in

the 5 →3 direction The other parental strand is copied only when a

sufficient stretch of its sequence has been exposed for DNA polymerase

to move along it in the 5 →3 mode Thus, one parental strand is copied

continuously to form the leading strand, while the other parental strand

is copied in an intermittent, or discontinuous, mode to yield a set of

Okazaki fragments that are joined later to give the lagging strand.

28.2 What Are the Properties of DNA Polymerases? All DNA

poly-merases share the following properties: (1) The incoming base is

se-lected within the DNA polymerase active site through base-pairing with

the corresponding base in the template strand, (2) chain growth is in

the 5 →3 direction antiparallel to the template strand, and (3) DNA

polymerases cannot initiate DNA synthesis de novo—all require a primer with a free 3 -OH to build upon DNA polymerase III

holoen-zyme, the enzyme that replicates of the E coli chromosome, is

com-posed of ten different kinds of subunits DNA polymerases are immobi-lized in replication factories

28.3 Why Are There So Many DNA Polymerases? Both prokaryotic and eukaryotic cells have a number of DNA polymerases These differ-ent enzymes can be assigned to families based on sequence similarities The various families of DNA polymerases fill different biological roles; the prominent roles include DNA replication, DNA repair, and telo-mere maintenance All DNA polymerases share a common architecture resembling a right hand, composed of distinct finger, thumb, and palm structural domains, each serving a specific role in the polymerase reaction.

28.4 How Is DNA Replicated in Eukaryotic Cells? Eukaryotic DNA is organized into chromosomes within the nucleus These chromosomes must be replicated once (and only once!) each cell cycle Progression through the cell cycle is regulated through checkpoints that control whether the cell continues into the next phase Cyclins and CDKs matain these checkpoints Replication licensing factors (MCM proteins) in-teract with origins of replication and render chromosomes competent for replication Three DNA polymerases—

genome replication DNA polymerase  initiates replication through

synthesis of an RNA DNA polymerase  is the principal DNA

polym-erase in eukaryotic DNA replication

28.5 How Are the Ends of Chromosomes Replicated? Telomeres are short, tandemly repeated, G-rich nucleotide sequences that form