Enzymes and DNA Polymerases

Enzymes Required for Replication

• Helicase unzips the DNA helix by breaking hydrogen bonds between nucleotides.
• Single strand binding proteins (SSBP) keeps strands separated during unzipping.
• Topoisomerase relieves the stress from unzipping by cutting one strand, unwinding, and reconnecting it.
• Primase binds to the origin of replication and synthesizes an RNA primer (5-10 nucleotides) complementary to the template.
• DNA polymerase can only attach nucleotides to existing nucleotides on the 3'-OH end, performing most of the DNA synthesis.
• DNA polymerase can only add a new nucleotide to the 3' end of an existing nucleotide.
• DNA polymerase and primase work together in a complex for priming.
• DNA polymerase epsilon (Pol ɛ) synthesizes the leading strand.
• DNA polymerase delta (Pol 6) synthesizes the lagging strand.

DNA Polymerases

• DNA Polymerase is responsible for most of the DNA replication, creating a polymer of DNA.
• DNA Polymerase replicates bacterial chromosomes at around 500 nucleotides per second.
• In human cells, replication occurs at about 50 nucleotides per second due to extensive proofreading mechanisms.
• E. coli has three DNA polymerases used for DNA synthesis.
• DNA polymerase I (Pol I) removes primers and replaces them with DNA on the lagging strand.
• DNA polymerase II (Pol II) is involved in DNA repair processes.
• DNA polymerase III (Pol III) is the main replication enzyme.
• All three DNA polymerases have 3'-to-5' exonuclease activity for proofreading, allowing removal of nucleotides when errors occur.
• DNA Pol I has 5'-to-3' exonuclease activity for removing RNA primers.

DNA Strands and Fragments

• Leading strand is synthesized continuously, moving toward the replication fork and adding to the 3' end.
• Lagging strand replicates moving away from the replication fork, forming Okazaki fragments.
• Each Okazaki fragment is made with a primase adding a primer, followed by DNA Polymerase III adding the DNA nucleotides.
• Primers are removed and refilled with DNA nucleotides by DNA Pol 1, and the backbone is joined by DNA ligase.

Chromosomes and DNA Structure

• A chromosome consists of a DNA molecule packed together with proteins.
• Bacterial chromosome is a double-stranded, circular DNA molecule with a small amount of protein.
• Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein.
• In bacteria, the DNA is "supercoiled" in a region called the nucleoid.
• Chromatin, a complex of DNA and protein, is in the nucleus of eukaryotic cells and fits through multilevel packing.
• During interphase, chromatin is organized into a 10-nm fiber, compacted into a 30-nm fiber through folding and looping.
• During interphase, chromosomes still occupy specific restricted regions in the nucleus.

Chromatin Packing and Organization

• Most chromatin is loosely packed during interphase and condenses prior to mitosis.
• Loosely packed chromatin is called euchromatin.
• During interphase, centromeres and telomeres are highly condensed into heterochromatin.
• Dense packing of heterochromatin makes it difficult for the cell to express genetic information.
• Histones can undergo chemical modifications that result in changes in chromatin organization.

Telomeres and Telomerase

• Linear chromosomes cannot replace the last primer removed from the 3' end of the lagging strand, leading to shortening with each division.
• Telomerase is an enzyme that synthesizes telomere repeat sequences at the ends of the strands.
• Telomerase uses an internal RNA template (not DNA itself) to do this.
• Telomeres are specialized structures at the ends of eukaryotic chromosomes, composed of specific repeat sequences.
• Telomeres protect chromosome ends from nucleases and maintain the integrity of linear chromosomes.
• Telomeres are not made by the replication complex.
• Their enzymes are similar to bacterial ones.

Telomerase Regulation and Function

• Telomerase activity is high in early development/childhood but low in most somatic adult cells.
• Exceptions to this include cells that must continue dividing, like lymphocytes.
• Telomerase plays a role in senescence (aging).
• Mice lacking telomerase activity had nonviable offspring after six generations.
• Telomerase contributes to cancer; cancer cells activate telomerase to maintain telomere length.

Mutations and Causes

• DNA damage occurs constantly due to random errors during replication, which is why DNA polymerases have proofreading mechanisms.
• External agents, like radiation and chemicals (mutagens), also cause mutations.
• Mutations are changes in the nucleotide base sequence of a genome.
• Mutations are rare and usually result in a nonfunctional gene product, but sometimes improve an organism's ability to survive.
• Genetic change is the driving force of evolution.
• Mutation can be spontaneous: a random change in the DNA arising from replication errors, or induced: resulting from exposure to mutagens.
• Physical or chemical agents disrupt DNA and are also known as carcinogens.
• Radiation such as UV light and X-rays can cause mutations.

Radiation-Induced Mutations

• Ionizing radiation (X-ray/gamma ray) causes electrons to excite, leading to covalent bond breakage of the sugar-phosphate backbone of DNA.
• Nonionizing radiation (UV) causes pyrimidine dimers (covalent bonding), warping the DNA shape.

Chemical Mutagens

• Nitrous acid is a chemical mutagen.
• Nucleotide analogs, which appear like normal nucleotides, can be erroneously substituted for nucleotides during replication.
• Nucleotide analogs can be useful as antiviral or anticancer drugs.

DNA Repair Mechanisms

• Mismatch repair removes incorrect bases and replaces them using the template strand as a guide.
• In E. coli, mismatch repair distinguishes new and old strands by looking at methylation; the new strand lacks methylation on certain sequences.
• UV light can cause thymine dimers, which are repaired by photoactivation/light repair through photolyase.
• The cell absorbs energy in the visible range to cut the thymine dimer.
• Excision repair is a nonspecific repair mechanism where damaged areas are recognized and removed by nucleases.
• The remaining gap is filled by DNA polymerase I and ligase.