BRS Genetics - R. Dudek (Lippincott)

3. Chromosome Replication

I. General Features

A. Chromosome replication occurs during S phase of the cell cycle and involves both DNA synthesis and histone synthesis to form chromatin.

B. The timing of replication is related to the chromatin structure. An inactive gene packaged as heterochromatin is replicated late in S phase (e.g., in a female mammalian cell, the inactive X chromosome called the Barr body is packaged as heterochromatin and is replicated late in S phase).

C. An active gene packaged as euchromatin is replicated early in S phase (e.g., in the pancreatic beta cell, the insulin gene will be replicated early in S phase. However, in other cell types (e.g., hepatocytes) where the insulin gene is inactive, it will be replicated late in S phase.

D. DNA polymerases absolutely require the 3′-OH end of a based paired primer strand as a substrate for strand extension. Therefore, a RNA primer (synthesized by a primase) is required to provide the free 3′-OH group needed to start DNA synthesis.

E. DNA polymerases copy a DNA template in the 3′ 5 direction, which produces a new DNA strand in the 5′ 3 direction.

F. Deoxyribonucleoside 5′-triphosphates (dATP, dTTP, dGTP, dCTP) pair with the corresponding base (A-T, G-C) on the template strand and form a 3′,5′-phosphodiester bond with the 3′-OH group on the deoxyribose sugar, which releases a pyrophosphate.

G. Replication is described as semiconservative which means that a molecule of double helix DNA contains one intact parental DNA strand and one newly synthesized DNA strand.

II. The Replication Process (Figure 3-1)

A. The process starts when topoisomerase nicks (or breaks) a single strand of DNA, which causes DNA unwinding.

B. Chromosome replication begins at specific nucleotide sequences located throughout the chromosome called replication origins. Eukaryotic DNA contains multiple replication origins to ensure rapid DNA synthesis. Normally, the S phase of the mammalian cell cycle is 8 hours.

C. DNA helicase recognizes the replication origin and opens up the double helix at that site, forming a replication bubble with a replication fork at each end. The stability of the replication fork is maintained by single-stranded binding proteins.

D. A replication fork contains a:

1.   Leading strand that is synthesized continuously by DNA polymerase δ (delta).

2.   Lagging strand that is synthesized discontinuously by DNA polymerase α (alpha). DNA primase synthesizes short RNA primers along the lagging strand. DNA polymerase α uses the RNA primer to synthesize DNA fragments called Okazaki fragments. Okazaki fragments end when they run into a downstream RNA primer. To form a continuous DNA strand from the Okazaki fragments, a DNA repair enzyme erases the RNA primers and replaces it with DNA. DNA ligase subsequently joins the all the DNA fragments together.

E. The anti-neoplastic drugs camptothecins (e.g., irinotecan, topotecan)anthracyclines (e.g., doxorubicin); epipodophyllotoxins (e.g., etoposide VP-16, teniposide VM-26); and amsacrine are topoisomerase inhibitors.

F. The anti-microbial drugs quinolones (e.g., ciprofloxacin, ofloxacin, levofloxacin, fluoroquinolones) are also topoisomerase inhibitors.

III. The Telomere

A. The human telomere is a 3-20 kb repeating nucleotide sequence (TTAGGG) located at the end of a chromosome. The 3-20 kb (TTAGGG)n array is preceded by 100-300 kb of telomere—associated repeats before any unique sequence is found.

B. The telomere allows replication of linear DNA to its full length. Because DNA polymerases cannot synthesize in the 3′ 5 direction or start synthesis de novo, removal of the RNA primers will always leave the 5′ end of the lagging strand shorter than the leading strand. If the 5′ end of the lagging strand is not lengthened, a chromosome would get progressively shorter as the cell goes through a number of cell divisions.

C. This problem of lagging strand shortening is solved by a special RNA-directed DNA polymerase or reverse transcriptase called telomerase (which has a RNA and protein component). The RNA component of telomerase carries a CCCUAA sequence (antisense sequence of the TTAGGG telomere) that recognizes the TTAGGG sequence on the leading strand and adds many repeats of TTAGGG to the leading strand.

D. After the repeats of TTAGGG are added to the leading strand, DNA polymerase α uses the TTAGGG repeats as a template to synthesize the complementary repeats on the lagging strand. Thus, the lagging strand is lengthened. DNA ligase joins the repeats to the lagging strand and a nuclease cleaves the ends to form double helix DNA with flush ends.

E. Telomerase is NOT utilized by a majority of normal somatic cells, so that chromosomes normally get successively shorter after each replication; this contributes to the finite lifespan of the cell.

F. Telomerase is utilized by stems cells and neoplastic cells so that chromosomes remain perpetually long. Telomerase may play a clinical role in aging and cancer.

IV. Types of DNA Damage and DNA Repair

A. Chromosomal breakage refers to breaks in chromosomes due to sunlight (or ultraviolet) irradiation, ionizing irradiation, DNA cross-linking agents, or DNA damaging agents. These insults may cause depurination of DNAdeamination of cytosine to uracil, or pyrimidine dimerization, which must be repaired by DNA repair enzymes.

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B. DNA repair involves DNA excision of the damaged site, DNA synthesis of the correct sequence, and DNA ligation. Some types of DNA repair use enzymes that do not require DNA excision.

C. The normal response to DNA damage is to stall the cell in the G1 phase of the cell cycle until the damage is repaired.

D. The system that detects and signals DNA damage is a multiprotein complex called BASC (BRCA1-associated genome surveillance complex). Some the components of BASC include: ATM (ataxia telangiectasia mutated) protein, nibrin, BRCA1 protein, and BRCA2 protein.

E. The clinical importance of DNA repair enzymes is illustrated by some rare inherited diseases that involve genetic defects in DNA repair enzymes such as xeroderma pigmentosa (XP), ataxia-telangiectasia, Fanconi anemia, Bloom syndrome, and hereditary nonpolyposis colorectal cancer.

F. Types of DNA damage include:

1.   Depurination. About 5,000 purines (A's or G's) per day are lost from DNA of each human cell when the N-glycosyl bond between the purine and deoxyribose sugar-phosphate is broken. This is the most frequent type of lesion and leaves the deoxyribose sugar-phosphate with a missing purine base.

2.   Deamination of cytosine to uracil. About 100 cytosines (C) per day are spontaneously deaminate to uracil (U). If the U is not corrected back to a C, then upon replication instead of the occurrence of a correct C-G base pairing and U-A base pairing will occur instead.

3.   Pyrimidine dimerization. Sunlight (UV radiation) can cause covalent linkage of adjacent pyrimidines forming for example, thymine dimers.

V. Summary Table of DNA Machinery (Table 3-1)

 

Figure 3-1. Replication fork. (A) A diagram of double helix DNA (Chromosome 1) at a replication origin (RO) site. DNA helicase (H) will bind at the RO and unwind the double helix into two DNA strands. This site is called a replication bubble (RB). At both ends of a replication bubble a replication fork (RF) forms. DNA synthesis occurs in a bidirectional manner from each RF (arrows). (B)Enlarged view of a RF at one end of the replication bubble. The leading strand serves as a template for continuous DNA synthesis in the 5′ 3 direction using DNA  polymerase δ (Pδ). The lagging strand serves as a template for discontinuous DNA synthesis in the 5′ 3 direction using DNA polymerase α (Pα). Note that DNA synthesis on the leading and lagging strands is in the 5′ 3 direction but physically are running in opposite directions. (C) DNA synthesis on the lagging strand proceeds differently than on the leading strand. DNA primase synthesizes RNA primers. DNA polymerase α uses these RNA primers to synthesize DNA fragments called Okazaki fragments (OF). Okazaki fragments end when they run into a downstream RNA primer. Subsequently, DNA repair enzymes remove the RNA primers and replace it with DNA. Finally, DNA ligase joins all the Okazaki fragments together.

 

Table 3-1 Summary of DNA Replication Machinery

Component

Function

Topoisomerase

Nicks (or breaks) a single strand of DNA which causes DNA unwinding

DNA helicase

Recognizes the replication fork and opens up the double helix

High Fidelity DNA-Directed DNA Polymerases

   DNA polymerase α

Synthesizes the lagging strand; 3′ 5 exonuclease absent*

   DNA polymerase β

Repairs DNA by base excision; 3′ 5 exonuclease absent

   DNA polymerase γ

Synthesizes mitochondrial DNA; 3′ 5 exonuclease present

   DNA polymerase δ

Synthesizes the leading strand; 3′ 5 exonuclease present; repairs DNA by nucleotide and base excision

   DNA polymerase ε

Repairs DNA by nucleotide and base excision; 3′ 5 exonuclease present

Low Fidelity DNA-Directed DNA Polymerases

   DNA polymerase ζ

Involved in hypermutation in B and T lymphocytes

   DNA polymerase η

Involved in hypermutation in B and T lymphocytes

   DNA polymerase ι

Involved in hypermutation in B and T lymphocytes

   DNA polymerase µ

Involved in hypermutation in B and T lymphocytes

RNA-Directed DNA Polymerase (Reverse Transcriptase)

   Telomerase

Lengthens the end of the lagging strand

   LINE 1/endogenous retrovirus reverse transcriptase

Converts RNA into cDNA, which can integrate elsewhere in the genome

Primase

Synthesizes short RNA primers

Ligase

Catalyzes the formation of the 3′,5′-phosphodiester bond; joins DNA fragments

Single-stranded binding proteins

Maintain the stability of the replication fork

High fidelity = DNA sequence faithfully copied; low fidelity = DNA sequence not faithfully copied (error prone)
* 3′
5 exonuclease = serves as proofreading activity

Review Test/Answers and Explanations

1. Human cells have a finite lifespan and this contributes to the aging process. Stem cells and neoplastic cells have indefinite life spans. The reason for these observations is that chromosomes in a cell get progressively shorter with each cell division because the telomere sequences at the ends of the chromosomes get shorter with each cell division. The chromosomes in stem cells and neoplastic cells do not generally shorten with each cell division. The enzyme utilized by stem cells and neoplastic cells to lengthen the telomeres is which of the following?

(A) DNA polymerase delta

(B) DNA polymerase alpha

(C) DNA ligase

(D) topoisomerase

(E) telomerase

1. The answer is (E). The other enzymes are involved in the replication process in general, but it is telomerase that can recognize the TTAGGG telomere sequence to that it can be replicated.

2. Some antineoplastic drugs act by inhibiting which of the following?

(A) DNA helicase

(B) topoisomerase

(C) telomerase

(D) DNA polymerase delta

(E) DNA polymerase alpha

2. The answer is (B). Many antineoplastic drugs act by inhibiting DNA replication.

3. Which one of the following is an accurate statement regarding chromosome replication?

(A) It is semiconservative.

(B) It occurs during G1 in the cell cycle.

(C) Inactive genes are replicated first.

(D) It starts with the synthesis of Okazaki fragments.

3. The answer is (A). Chromosome replication occurs during the S phase of the cell cycle. It starts when topoisomerase breaks a single strand of DNA, which causes DNA unwinding. Active genes are replicated early in S and inactive genes late in the S phase. Replication is semiconservative because there is one intact parental strand in a double helix of DNA and a newly synthesized strand.

4. The leading strand of DNA in the replication fork is synthesized by which one of the following mechanisms?

(A) continuously by DNA polymerase alpha.

(B) discontinuously by DNA polymerase delta.

(C) continuously by DNA polymerase delta.

(D) discontinuously by DNA polymerase alpha.

4. The answer is (C). The lagging strand of DNA is synthesized discontinuously by DNA polymerase alpha, which synthesizes Okazaki fragments, which then have to be joined by a DNA ligase. The leading strand of DNA is synthesized continuously by DNA polymerase delta.

5. The autosomal recessive disease Fanconi anemia is characterized by chromosome breakage and rearrangements and most individuals with the disease will develop some kind of cancer. Which one of the following is defective in individuals with Fanconi anemia?

(A) DNA polymerase delta

(B) DNA repair enzyme

(C) DNA ligase

(D) DNA primase

5. The answer is (B). In Fanconi anemia, DNA damage goes unrepaired and eventually reaches a point where the chromosome is unstable. The result is that there is chromosome breakage with rearrangements of chromosomal material. When a break occurs in a tumor suppressor gene, or proto-oncogenes are activated by chromosome rearrangements, the development of a malignancy is likely.