2.

DNA Replication, Mutation, Repair

a). DNA replication i). Cell cycle/ semi-conservative replication ii). Initiation of DNA replication iii). Discontinuous DNA synthesis iv). Components of the replication apparatus b). Mutation i). Types and rates of mutation ii). Spontaneous mutations in DNA replication iii). Lesions caused by mutagens c). DNA repair i). Types of lesions that require repair ii). Mechanisms of repair Proofreading by DNA polymerase Mismatch repair Excision repair iii). Defects in DNA repair or replication

The mammalian cell cycle

Rapid growth and preparation for DNA synthesis DNA synthesis and histone synthesis phase

G0
Quiescent cells

G1
phase

The S (for synthesis) phase of the cell cycle is when chromosomes are replicated. This requires DNA synthesis and histone synthesis (the latter to make the proteins that will package the newly replicated DNA).

phase

phase

G2

Growth and preparation for cell division

Mitosis

DNA replication is semi-conservative

Parental DNA strands

Each of the parental strands serves as a template for a daughter strand

Daughter DNA strands

DNA replication is said to be "semi-conservative" because each strand of the DNA double helix serves as a template for the synthesis of a new complementary DNA strand. Hence, the newly replicated chromosome consists of one old and one new DNA strand

Origins of DNA replication on mammalian chromosomes

Each chromosome consists of a very long DNA strand, the complete length of which needs to be replicated in a relatively short (a few hours) period of time. This is accomplished by having multiple origins of DNA replication on each chromsome, which are spaced every ~150 kb. Replication initiates independently at each origin and proceeds bidirectionally as the new DNA strands (red) are synthesized. The resulting "replication bubbles" then fuse together or merge completing the synthesis of the daughter chromosomes.

Origins of DNA replication on mammalian chromosomes

origins of DNA replication (every ~150 kb) 5 3 3 5

bidirectional replication

replication bubble fusion of bubbles

5 3
5 3

daughter chromosomes

3 5
3 5

Initiation of DNA synthesis at the E. coli origin (ori)

The single, circular E. coli chromosome has one origin of replication (ori). Initiation of replication begins with the binding of dnaA proteins to the ori sequence. As these proteins coalesce, the adjacent DNA is forced to undergo melting into single strands. This then allows the dnaB and dnaC proteins to bind the single-stranded DNA and further unwind the double helix, catalyzed by the dnaB protein which is a DNA "helicase" or DNA unwinding enzyme.

Initiation of DNA synthesis at the E. coli origin (ori)

origin DNA sequence

5 3

3 5

binding of dnaA proteins

A A A

A
A

DNA melting induced by the dnaA proteins

dnaA proteins coalesce

dnaB and dnaC proteins bind to the single-stranded DNA

A
A A

A
A

B C

dnaB further unwinds the helix

 As further unwinding occurs, which displaces the dnaA proteins, the dnaG protein binds. This protein is a "primase" and synthesizes a short RNA primer of about 5 nucleotides. Because the primase synthesizes an RNA strand, it is an RNA polymerase. The primer provides a free 3' OH end to initiate DNA synthesis. As a rule, DNA polymerases cannot initiate DNA synthesis de novo, but can only add onto an existing 3' OH. Hence, the need for the RNA primer. In contrast to DNA polymerases, RNA polymerases can initiatate synthesis de novo.

dnaG (primase) binds...

A A A

B C
A

dnaB further unwinds the helix and displaces dnaA proteins

A A A A A A

...and synthesizes an RNA primer

G
RNA primer

B C

The "primasome" consists of the dnaB, dnaC, and dnaG proteins.

B C

Primasome dna B (helicase) dna C dna G (primase)

template strand 3 OH 5 RNA primer (~5 nucleotides)

DNA polymerase
5 5 RNA primer 3

5 newly synthesized DNA

Once the RNA primer has been synthesized, DNA polymerase can then bind and begin to synthesize DNA. DNA polymerase catalyzes an attack by the 3' OH on the alpha phosphate of the dGTP, forming a 3', 5'-phosphodiester bond, and releasing pyrophosphate, which is then hydrolyzed to two molecules of inorganic phosphate. All DNA polymerases require a primer (or a growing DNA chain) with a free 3' OH. The new strand of DNA grows in a 5' to 3' direction; however, some DNA polymerases also have a 3' to 5' proofreading activity, which can remove the 3' terminal nucleotide in case the polymerase makes a mistake.

Discontinuous synthesis of DNA

Because DNA synthesis always proceeds in a 5' to 3' direction, and because the two DNA strands are arranged antiparallel with respect to each other, only one of the two newly synthesized strands can be made "continuously" (continuous red line) as the DNA polymerase moves away from the origin of replication and more DNA template is exposed. The other strand has to be made "discontinuously" in short pieces (short red lines). This latter strand is called the "lagging strand" while the continuously synthesized strand is called the "leading strand."

Discontinuous synthesis of DNA

5 3

3 5

5 3

3 5

5 3

3 5

Each replication fork has a leading and a lagging strand

leading strand (synthesized continuously)

replication fork 5 3 3 5

replication fork

5 3
3 5

5 3

3 5

The small pieces of DNA that comprise the lagging strand are called "Okazaki fragments." They are eventually ligated together forming a continuous DNA strand.

lagging strand (synthesized discontinuously) The leading and lagging strand arrows show the direction of DNA chain elongation in a 5 to 3 direction The small DNA pieces on the lagging strand are called Okazaki fragments (100-1000 bases in length)

RNA primer direction of leading strand synthesis 3 5 replication fork 5 3

The next series of figures shows the process of DNA synthesis at the so-called "replication fork." Note the direction (arrows) of leading strand synthesis and lagging strand synthesis (both are in a 5' to 3' direction). As stated previously, DNA polymerase moves continuously along the leading strand. As the template DNA unwinds, exposing the single-stranded template for the lagging strand, primase has to synthesize an RNA primer to which the DNA polymerase synthesizing the lagging strand can bind.

3 5

direction of lagging strand synthesis

Strand separation at the replication fork causes positive supercoiling of the downstream double helix

The strand separation process (unwinding the complementary WatsonCrick DNA strands) causes overwinding ahead of the fork. Any DNA that is overwound (or underwound) is said to be supercoiled. Overwound DNA is positively supercoiled. The increasing torsional stress needs to be dissipated in order for the fork to continue to unwind so that replication can proceed. This is accomplished by DNA topoisomerases, which cut the DNA strands, unwind them and reseal the strands. As they do so they introduce negative supercoiling into the DNA to compensate for the positive supercoiling. Gram-negative bacteria, such as E. coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, can be killed by fluoroquinolone antibiotics, which inhibit DNA gyrase, a topoisomerase II. Topoisomerase II cuts both strands of DNA, swivels them and rejoins them. Topoisomerase I cuts only one strand, and relaxes negative supercoils.

Strand separation at the replication fork causes positive supercoiling of the downstream double helix 3 5 5 3 3 5

DNA gyrase is a topoisomerase II, which breaks and reseals the DNA to introduce negative supercoils ahead of the fork Fluoroquinolone antibiotics target DNA gyrases in many gram-negative bacteria: ciprofloxacin and levofloxacin (Levaquin)

Movement of the replication fork

5 3

As the replication fork moves further to the left, opening up more DNA, another RNA primer has to be synthesized.

Movement of the replication fork

Each RNA primer (dashed red line) on the lagging strand then serves as a starting point for the initiation of DNA synthesis (Okazaki fragment; solid red line).

RNA primer Okazaki fragment

RNA primer

 E. coli DNA polymerase III initiates at the RNA primer, synthsizing DNA to fill in the gap up to the next RNA primer. However, it falls off the template DNA once it reaches the next RNA primer. At this point, DNA polymerase I takes over. It contains a 5' to 3' exonuclease activity that can remove the RNA primer while it simultaneously adds DNA nucleotides to the 3' end of the Okazaki fragment.

RNA primer 5

pol III 5 DNA polymerase III initiates at the primer and elongates DNA up to the next RNA primer

newly synthesized DNA (100-1000 bases) (Okazaki fragment) 5

pol I

DNA polymerase I inititates at the end of the Okazaki fragment and further elongates the DNA chain while simultaneously removing the RNA primer with its 5 to 3 exonuclease activity

newly synthesized DNA (Okazaki fragment)

DNA ligase seals the gap by catalyzing the formation of a 3, 5-phosphodiester bond in an ATP-dependent reaction 3

5
However, DNA polymerase I cannot seal the gap between the two adjacent Okazaki fragments. This job is carried out by DNA ligase, which catalyzes the formation of a 3', 5'-phosphodiester bond in an ATP-dependent reaction. Thus, it takes one RNA polymerase (primase), two DNA polymerases (III and I), and DNA ligase to synthesize the lagging strand. Once initiation occurs on the RNA primer, the leading strand only requires DNA polymerase III for its synthesis.

Proteins at the replication fork in E. coli

This figure shows the proteins required for DNA synthesis at the replication fork. In addition to the proteins required for leading and lagging strand synthesis, there are several others that act upstream of the replication fork. The act of unwinding the DNA double helix puts torsional stress in the form of positive supercoils in the DNA upstream of the fork. To overcome this, DNA gyrase, which is a topoisomerase II, breaks and reseals the DNA in order to introduce negative supercoils in the DNA, thus overcoming the positive supercoils. The unwinding itself is carried out by the Rep protein, which is a helicase. Finally, to keep the unwound strands single-stranded, they bind SSB (single-strand binding protein).

Proteins at the replication fork in E. coli

Rep protein (helicase)
pol III 3 5

5 3

G
Single-strand binding protein (SSB)

Primasome

DNA ligase

C B

pol III

DNA gyrase - this is a topoisomerase II, which breaks and reseals double-stranded DNA to introduce negative supercoils ahead of the fork

pol I

Components of the replication apparatus

dnaA Primasome dnaB dnaC dnaG DNA gyrase binds to origin DNA sequence helicase (unwinds DNA at origin) binds dnaB primase (synthesizes RNA primer) introduces negative supercoils ahead of the replication fork helicase (unwinds DNA at fork) binds to single-stranded DNA primary replicating polymerase removes primer and fills gap seals gap by forming 3, 5-phosphodiester bond

Rep protein SSB DNA pol III DNA pol I DNA ligase

This table lists the proteins required for replication of the E. coli chromosome, and their activities.

Properties of DNA polymerases

The main DNA polymerases required for DNA replication in E. coli are DNA polymerases I and III. They both have 5' to 3' polymerizing activity and 3' to 5' proofreading activity. DNA polymerase I also removes the RNA primer and is also a DNA repair enzyme, and thus requires a 5' to 3' exonuclease activity. In contrast to just three DNA polymerases in E. coli, human cells have at least five DNA polymerases. The enzymes thought to be responsible for replication of nuclear DNA are DNA polymerases alpha, delta, and epsilon. Alpha is associated with an RNA primase and it is thought that these activities are responsible for synthesizing a short RNA-DNA primer. There is some uncertainty as to the function of alpha in lagging strand synthesis. Since it seems to lack a 3 to 5 exonuclease activity, it may not be able to synthesize DNA with high fidelity and thus it may not be the main lagging strand polymerase. If not, the main polymerase for the lagging strand may be delta, which is also specifically responsible for synthesis of the leading strand. DNA polymerase epsilon may function like the bacterial DNA polymerase I, by removing primers and extending the Okazaki fragment. In humans, there is also a requirement for DNA ligase. Proliferating cell nuclear antigen (PCNA) has a role in both replication and repair. It is a toroidal-shaped (donut) protein which encircles DNA and can slide bidirectionally along the duplex. One of its functions is to serve as a processivity factor for DNA polymerase delta and epsilon. PCNA holds the polymerase to the DNA template for rapid and processive DNA synthesis. Recently, it has been discovered that PCNA also interacts with proteins involved in cellcycle progression.

Properties of DNA polymerases DNA polymerases of E. coli_ Polymerization: 5 to 3 Proofreading exonuclease: 3 to 5 Repair exonuclease: 5 to 3 pol I yes yes yes pol II yes yes no pol III (core) yes yes no

DNA polymerase III is the main replicating enzyme DNA polymerase I has a role in replication to fill gaps and excise primers on the lagging strand, and it is also a repair enzyme and is used in making recombinant DNA molecules

all DNA polymerases require a primer with a free 3 OH group

all DNA polymerases catalyze chain growth in a 5 to 3 direction some DNA polymerases have a 3 to 5 proofreading activity

Mutation

Types and rates of mutation

Type Genome mutation Mechanism chromosome missegregation (e.g., aneuploidy) chromosome rearrangement (e.g., translocation) base pair mutation (e.g., point mutation, or small deletion or insertion Frequency________ 10-2 per cell division

Chromosome mutation

6 X 10-4 per cell division

Gene mutation

10-10 per base pair per cell division or 10-5 - 10-6 per locus per generation

Mutation rates* of selected genes

Gene New mutations per 106 gametes 6 2.5 43 32 2 44 60 5 to 40 to 5 to 105 to 57 to 3 to 100 to 120 to 12

Achondroplasia Aniridia Duchenne muscular dystrophy Hemophilia A Hemophilia B Neurofibromatosis -1 Polycystic kidney disease Retinoblastoma

*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

CATTCACCTGTACCA GTAAGTGGACATGGT

normal sequence

CATCCACCTGTACCA GTAGGTGGACATGGT

transition (T-A to C-G)

CATGCACCTGTACCA GTACGTGGACATGGT

transversion (T-A to G-C)

base pair substitutions transition: pyrimidine to pyrimidine transversion: pyrimidine to purine

CATCACCTGTACCA GTAGTGGACATGGT

deletion

CATGTCACCTGTACCA GTACAGTGGACATGGT

insertion

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 C A

C G
C A

replication of C-G should give daughter strands each with C-G

tautomer formation C during replication will result in mispairing and insertion of an improper A in one of the daughter strands

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

Attack by oxygen free radicals leading to oxidative damage

many different oxidative modifications occur by smoking, etc. 8-oxyG causes G to T transversions O
H N O NH N NH2 NH

O
N NH NH N NH2

guanine

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 Altered base Incorrect base Acid and heat depurination (~104 purines per day per cell in humans) Ionizing radiation; alkylating agents Spontaneous deaminations cytosine to uracil adenine to hypoxanthine

Deletion-insertion
Dimer formation Strand breaks Interstrand cross-links Tautomer formation

Intercalating reagents (acridines)

UV irradiation Ionizing radiation; chemicals (bleomycin) Psoralen derivatives; mitomycin C Spontaneous and transient

Mechanisms of Repair
Mutations that occur during DNA replication are repaired when possible by proofreading by the DNA polymerases
Mutations that are not repaired by proofreading are repaired by mismatch (post-replication) repair followed by excision repair Mutations that occur spontaneously any time are repaired by excision repair (base excision or nucleotide excision)

Mismatch (post-replication) repair

(reduces DNA replication errors 1,000-fold)

the parental DNA strands are methylated on certain adenine bases

CH3

CH3

5 3
CH3

mutations on the newly replicated strand are identified by scanning for mismatches prior to methylation of the newly replicated DNA

the mutations are repaired by excision repair mechanisms after repair, the newly replicated strand is methylated

CH3

deamination

Excision repair

ATGCUGCATTGA TACGGCGTAACT
uracil DNA glycosylase thymine dimer

ATGC GCATTGA TACGGCGTAACT

repair nucleases

ATGCUGCATTGATAG TACGGCGTAACTATC
excinuclease

AT GCATTGA TACGGCGTAACT
DNA polymerase b

AT (~30 nucleotides) AG TACGGCGTAACTATC

DNA polymerase b

ATGCCGCATTGA TACGGCGTAACT
DNA ligase

ATGCCGCATTGATAG TACGGCGTAACTATC
DNA ligase

ATGCCGCATTGA TACGGCGTAACT
Base excision repair

ATGCCGCATTGATAG TACGGCGTAACTATC
Nucleotide excision repair

Deamination of cytosine can be repaired

cytosine

uracil

Deamination of 5-methylcytosine cannot be repaired

5-methylcytosine

thymine

More than 30% of all single base changes that have been detected as a cause of genetic disease have occurred at 5-mCpG-3 sites

Correlation between DNA repair activity in fibroblast cells from various mammalian species and the life span of the organism 100

human

elephant
cow Life span
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 Werners syndrome caused by mutations in a DNA helicase gene premature aging