DNA and The Gene: Synthesis and Repair

What Are Genes Made Of?

  • Chromosomes are comprised of DNA and protein.
  • Initially, it was unknown whether genes were comprised of DNA or protein.
  • The consensus supported the hypothesis that genes were comprised of proteins.

DNA’s Primary Structure

  • The primary structure of DNA has two major components:
    1. A backbone made up of the sugar and phosphate groups of deoxyribonucleotides.
    2. A series of nitrogen-containing bases that project from the backbone.

DNA’s Primary Structure: Directionality

  • DNA has directionality.
  • One end has an exposed hydroxyl group on the 3' carbon of deoxyribose.
  • The other end has an exposed phosphate group on a 5' carbon.
  • The molecule thus has a 3' end and a 5' end.

DNA’s Secondary Structure

  • Watson and Crick proposed:
    • Two DNA strands line up in the opposite direction to each other (antiparallel fashion).
    • The antiparallel strands twist to form a double helix.
  • The secondary structure is stabilized by complementary base pairing:
    • Adenine (A) hydrogen bonds with thymine (T).
    • Guanine (G) hydrogen bonds with cytosine (C).

How Do the Old and New DNA Strands Interact?

  • Semiconservative replication:
    • The parental DNA strands separate at the replication fork.
    • Each parental DNA strand is used as a template for the synthesis of a new strand.
    • Each daughter cell consists of one old and one new strand.

DNA Polymerase: Enzyme That Synthesizes DNA

  • DNA polymerase can add bases to only the 3' end of a growing DNA chain.
  • DNA synthesis always proceeds in the 5' → 3' direction.

DNA Polymerase: Energy Source

  • DNA polymerization is exergonic (energy-releasing reactions).
  • Monomers that act as substrates (reactants) are deoxyribonucleoside triphosphates (dNTPs): ATP, TTP, GTP, CTP.
  • dNTPs have high potential energy because of their three closely packed phosphate groups.

How Does DNA Replication Get Started?

  • Prokaryotes have bidirectional DNA replication.
  • In bacterial chromosomes, the replication process:
    • Begins at a single location (origin of replication – site where DNA replication starts) because prokaryotic chromosomes are circular.
  • Eukaryotes also have bidirectional replication but have multiple origins of replication (because eukaryotes have linear chromosomes).
  • A replication fork is the Y-shaped region where the DNA is split into two separate strands for copying.

How Is the Helix Opened and Stabilized?

  • Several proteins are responsible for opening and stabilizing the double helix:
    • Enzyme helicase catalyzes the breaking of hydrogen bonds between the two DNA strands to separate them.
    • Single-strand DNA-binding proteins (SSBPs) attach to the separated strands to prevent them from closing.
  • Enzyme topoisomerase cuts and rejoins the DNA:
    • Downstream of the replication fork, relieving tension in the helix.

How Is the Leading Strand Synthesized?

  • DNA polymerase requires a primer (a few nucleotides bonded to the template strand) that provides a free 3' hydroxyl (OH) group for phosphodiester bond formation with an incoming dNTP.
  • Primase (a type of RNA polymerase) synthesizes a short RNA segment that serves as a primer. DNA polymerase III then adds bases to the 3' end of the primer.
  • The product is called the leading strand, or continuous strand. It leads into the replication fork and is synthesized continuously in the 5' → 3' direction.

The Lagging Strand

  • The other DNA strand is called the lagging strand.
  • It is synthesized discontinuously, in the direction away from the replication fork, because DNA synthesis must proceed in the 5' → 3' direction.

How Is the Lagging Strand Synthesized?

  • Synthesis of the lagging strand starts when primase synthesizes a short stretch of RNA, acting as a primer. DNA polymerase III then adds bases to the 3' end of the primer.
  • DNA polymerase moves away from the replication fork, while helicase continues to open the replication fork and expose single-stranded DNA on the lagging strand.

The Discontinuous Replication Hypothesis

  • Once primase synthesizes an RNA primer on the lagging strand, DNA polymerase might synthesize short fragments of DNA along the lagging strand. These fragments would later be linked together to form a continuous whole strand.
  • This hypothesis was tested by Okazaki and his colleagues.

The Discovery of Okazaki Fragments

  • The lagging strand is synthesized as short discontinuous fragments called Okazaki fragments.
  • DNA polymerase I removes the RNA primer at the beginning of each Okazaki fragment and fills in the gap.
  • The enzyme DNA ligase joins the Okazaki fragments to form a continuous DNA strand.
  • Because Okazaki fragments are synthesized independently and joined together later, the lagging strand is also called the discontinuous strand.

Replicating the Ends of Linear Chromosomes

  • Telomere: Region at the end of a linear chromosome that does not contain genes and consists of short, repeating stretches of bases.
  • Replication of telomeres can be problematic: leading-strand synthesis results in a normal copy of the DNA molecule, but the telomere on the lagging strand shortens during DNA replication.

Replicating the Ends of Linear Chromosomes: The Problem

  • Replication fork reaches the end of a linear chromosome: there is no way to replace the RNA primer from the lagging strand with DNA because there is no available primer for DNA synthesis.
  • The primer is removed, leaving a section of single-stranded DNA (lagging strand) at one end of each new chromosome.
  • Remaining single-stranded DNA is eventually degraded, resulting in shortening of the chromosome.

Replicating the Ends of Linear Chromosomes: Telomerase

  • The enzyme telomerase adds more repeating bases to the end of the lagging strand, catalyzing the synthesis of DNA from an RNA template carried with it.
  • Primase then makes an RNA primer, and DNA polymerase uses the primer to synthesize the lagging strand. Ligase connects the new sequence, preventing the lagging strand from getting shorter with each replication.

Replication in Somatic Cells

  • Somatic cells normally lack telomerase, so chromosomes of somatic cells progressively shorten as the individual ages.
  • Hypothesis: Telomere shortening has a role in limiting the amount of time cells remain in an actively growing state.

Repairing Mistakes and DNA Damage

  • DNA replication is very accurate, with an average error rate of less than one mistake per billion bases.
  • DNA polymerase is highly selective in matching complementary bases correctly, inserting the incorrect base only about once every 100,000 bases added.
  • Repair enzymes remove defective bases and repair them if mistakes remain after synthesis is complete or if DNA is damaged.

How Does DNA Polymerase Proofread?

  • DNA polymerase can proofread its work by checking the match between paired bases and correcting mismatched bases when they do occur.
  • If the enzyme finds a mismatch, it pauses and removes the mismatched base that was just added.
  • DNA polymerase III can do this because its ee (epsilon) subunit acts as an exonuclease, removing deoxyribonucleotides from DNA.
  • This proofreading process reduces the error rate to about 1×10−71 \times 10^{-7}.

How Does DNA Polymerase Proofread? Mismatch Repair

  • If DNA polymerase leaves a mismatched pair behind in the newly synthesized strand, a battery of enzymes springs into action to correct the problem, in spite of its proofreading ability.
  • Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete.
  • Mismatch repair enzymes recognize the mismatched pair, remove a section of the newly synthesized strand that contains the incorrect base, and fill in the correct bases.

Repairing Damaged DNA

  • DNA can be broken or altered by various chemicals and types of radiation.
  • UV light can cause thymine dimers to form, producing a kink in the DNA strand.

Nucleotide Excision Repair

  1. Error detection.
  2. Nucleotide excision.
  3. Nucleotide replacement.
  4. Nucleotide linkage.

Xeroderma Pigmentosum: A Case Study

  • Xeroderma pigmentosum (XP) is a rare autosomal recessive disease in humans, characterized by the development of skin lesions.
  • XP is caused by mutations of one of several nucleotide excision repair systems. The cells of people with XP cannot repair DNA damaged by ultraviolet radiation.

DNA Repair Genes and Cancer

  • Defects in the genes required for DNA repair are frequently associated with cancer.
  • If mutations in the genes involved in the cell cycle go unrepaired, the cell may begin to grow in an uncontrolled manner, resulting in the formation of a tumor.
  • If the overall mutation rate in a cell is elevated because of defects in DNA repair genes, then the mutations that trigger cancer become more likely.