Organization, Synthesis, and Repair of DNA

Chapter 11 Organization, Synthesis, and Repair of DNA



I. DNA Organization
A. Overview

The DNA helix is composed of two strands that are antiparallel and complementary.


1. Two characteristics of DNA that affect its organization in the nucleus: extreme length and predominance of noncoding sequences

DNA: predominance of noncoding sequences; 98% of the genome


2. Supercoiling allows compaction of DNA while allowing selective expression of genetic information.

3. More than 98% of the genome is noncoding:
a. Introns divide the coding regions (exons).

b. Regulation of gene expression as promoters and enhancers (or silencers)

c. Pseudogenes

d. Repetitive DNA

B. Nucleosomes
1. Chromatin is a complex of double-strand DNA (Fig. 11-1) associated with histone proteins and other proteins that folds and packs to form the chromosomes.
image

11-1 DNA double helix. Strands are complementary (i.e., A is always paired with T, and G is paired with C) and antiparallel (i.e., one strand runs in the 3′ → 5′ direction; the other runs in the 5′ → 3′). Adenine-thymine (A-T) pairs have two hydrogen bonds; guanine-cytosine (G-C) pairs have three hydrogen bonds.



Basic histones bind to acidic DNA


a. Histones are basic proteins (positively charged) that associate tightly with acidic DNA (negatively charged).

Antibodies against histones are increased in drug-induced lupus erythematosus.


Nucleosome: DNA wrapped twice around histone core; connected by linker DNA


b. The nucleosome, the basic structural unit of chromatin, is made up of a core containing a total of eight histones (two molecules each of histones H2A, H2B, H3, and H4), around which DNA is wrapped twice (Fig. 11-2).

c. Linker DNA, which is associated with a fifth type of histone (H1), connects adjacent nucleosomes and produces the “beads on a string” form of chromatin.
image

11-2 Schematic model depicting the organization of human DNA. A and B, Double-strand DNA wraps around histone cores to form nucleosomes, which are connected by intervening linker DNA to which another histone is bound. C, Supercoiling produces a more compact 30-nm chromatin fiber. D, In interphase chromosomes, long stretches of 30-nm chromatin loop out from a protein scaffold. E and F, Further folding of the scaffold yields the highly condensed structure of a metaphase chromosome.



Chromatin fibers: nucleosomes arranged in supercoils


d. Supercoiling, mediated by histone H1, produces the more compact 30-nm chromatin fiber.

Chromosomes: compaction of chromatin fibers


e. Higher-order coiling and folding of chromatin permits compaction of DNA into chromosomes.

Fluoroquinolone antibiotics inhibit DNA compaction and inhibit gyrase.


(1) Fluoroquinolone antibiotics, such as ciprofloxacin, which block DNA compaction by inhibiting bacterial DNA gyrase (topoisomerase II), have little effect on the equivalent human enzyme.

C. Pseudogenes
1. A pseudogene is produced by a retrovirus that makes a DNA copy of a messenger RNA.

2. Pseudogenes cannot be expressed because they lack a promoter and introns.

Pseudogenes: DNA copy of a messenger RNA; lack promoter and introns


3. Pseudogenes are replicated along with the genome at cell division, but they remain dormant and have no ability to be expressed.

D. Repetitive DNA and transposons
1. Repetitive DNA is constituted from repeated sequences (from two base pairs to several thousand) that are arranged in tandem; they constitute about 30% of the genome.

Repetitive DNA: repeated sequences arranged in tandem; 30% of the genome


2. An alternate term for repetitive DNA is satellite DNA (i.e., appears as a satellite band in centrifugation).

3. Unequal crossover events produce repetitive DNA.

4. Transposons, also referred to as jumping genes, are DNA sequences that can jump to different areas of the genome within the same cell.

Transposons: jumping genes; insert themselves into the genome


Retrotransposons: cellular reverse transcriptase; DNA inserts created from mRNA; inactivates genes


5. Retrotransposons are a class of jumping genes that use reverse transcription by a cellular reverse transcriptase (see Section II, E later) encoded in the genome of normal, uninfected cells.
a. Insertion of a jumping gene into an active gene causes an insertion mutation, which prevents expression of normal protein.

b. Jumping gene insertion can cause genetic diseases such as hemophilia and Duchenne muscular dystrophy.

c. The most abundant type of retrotransposon in the human genome retrotransposons is the L1 family.

II. DNA Synthesis
A. Overview
1. Two signals initiate DNA synthesis: cell division and DNA repair.

2. DNA must be physically exposed to the enzymes and substrates for replication or repair.

3. DNA repair and DNA replication occur at distinct times during the cell cycle.

B. The cell cycle (Fig. 11-3)
1. The cell cycle is an ordered sequence of events during which proliferating cells replicate their chromosomes and separate into daughter cells.

2. The cell cycle consists of interphase, followed by mitosis (nuclear replication) and cytokinesis (cell division).
a. Interphase consists of a G1 (first gap or growth) phase, an S (synthesis) phase, and a G2 phase.
image

11-3 Cell cycle in eukaryotic cells. DNA synthesis occurs only during the synthesis (S) phase. The gap or growth (G) phases are periods of growth of organelles and mitotic structures. Anticancer drugs exert their effect at different stages, depending on their mode of action. Damage to DNA or improper spindle formation leads to arrest, preventing entry to the next stage.



G1 to S phase is the most important phase to control.


G1 and G2 phases: gap (G) periods of RNA and protein synthesis


(1) The G1 and G2 phases are periods of RNA and protein synthesis for general growth of organelles and mitotic structures; no DNA synthesis occurs.

Drugs that inhibit the G2 phase, in which tubulin is synthesized, include etoposide and bleomycin.


(2) Resting cells in the G0 phase reenter the cell cycle early in the G1 phase, the most variable phase of the cell cycle.

S phase: DNA synthesis


(3) Replication of DNA occurs during the S phase of the cycle. During mitosis and cytokinesis of the M phase, replicated chromosomes align on the mitotic spindle and then segregate evenly to the daughter cells.

Cell cycle: interphase, consisting of G1, S, and G2 phases, followed by mitosis and cytokinesis


3. Cell cycle control proteins regulate progression through the cell cycle.
a. Cyclin-dependent protein kinases (CDKs) phosphorylate various target proteins, thereby modulating their activity at different stages of the cell cycle.

CDKs control entry into stages of the cell cycle.


Cyclins activate CDKs, and concentrations change during the cell cycle.


b. Cyclins activate CDKs by binding to them. Concentrations of various cyclins rise and fall throughout the phases of the cell cycle, leading to formation of specific cyclin-CDK complexes that control entry into the S phase and entry into and exit from mitosis.

Arrests at checkpoints prevent abnormalities and permit repair of damaged DNA.


4. Checkpoints are positions in the cell cycle where progress can be arrested to prevent formation of abnormal daughter cells (see M, G1, and G2 arrests) (see Fig. 11-3).
a. When DNA is damaged, the G1 checkpoint prevents entry into the S phase, and the G2 checkpoint prevents entry into mitosis.

TP53 is a tumor-suppressor gene required for DNA repair at G1 and G2 checkpoints.


(1) Expression of TP53, a tumor-suppressor gene (on chromosome 17) that is required for operation of the G1 and G2 checkpoints, codes for a protein product that inhibits activated CDKs.

(2) Arrest of the cell cycle allows time for DNA repair and reduces the chances for perpetuation of mutations.

(3) Inactivation of the TP53 suppressor gene is seen in most human cancers.

M checkpoint prevents abnormal chromosome numbers from nondisjunction.


b. The M checkpoint prevents exit from mitosis when the spindle is abnormal, thereby preventing dysfunctional segregation of chromatids and formation of daughter cells with an abnormal number of chromosomes.

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Jun 18, 2016 | Posted by in BIOCHEMISTRY | Comments Off on Organization, Synthesis, and Repair of DNA

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