5+Replication+and+Proofreading+Short

Replication Overview

• Fundamental process of copying DNA to ensure genetic information is passed on during cell division.

DNA Replication Basics

• DNA double-helix unwound in specific region (replication fork).

• Template strands: Each DNA strand serves as a template for the synthesis of new complementary strands.

DNA Replication Mechanisms

• Nucleotide Addition: Individual deoxyribonucleoside triphosphates added sequentially to growing strands.

• Base Pairing: The order of base addition determined by complementary pairing to the template strand.

• The result is two new semiconservative double strands.

  DNA Replication Mechanism

  1. Unwinding DNA: The process begins at the replication fork, where DNA helicase unwinds and separates the double-stranded DNA, creating single-stranded regions accessible for replication.

  2. Template Strand Recognition: Each separated DNA strand serves as a template. DNA polymerases recognize and bind to the single-stranded template, which provides the necessary sequence information for the synthesis of new strands.

  3. Nucleotide Addition: Individual deoxyribonucleoside triphosphates are added one by one to the growing DNA strand. This addition occurs through a polymerization reaction where the incoming nucleotide's triphosphate releases two phosphates, providing energy for the bond formation.

  4. Base Pairing: The order of nucleotide addition follows the complementary base pairing rules (A with T, and C with G). This specificity ensures that the new strand is an exact copy of the original strand.

  5. Formation of New Strands: The result is the formation of two new double-stranded DNA molecules, each comprising one original template strand and one newly synthesized strand. This semiconservative method ensures accurate genetic replication.

Key Players in DNA Replication

DNA Helicase

• Function: Unwinds and separates DNA strands, facilitating the replication process.

• Mechanism: Binds and hydrolyzes ATP, leading to conformational changes that pry apart the DNA strands.

Single-Strand Binding Proteins (SSBs)

• Prevents re-annealing of separated strands, ensuring accessibility for replication enzymes.

DNA Primase

• Role: Synthesizes short RNA primer (10-200 nucleotides) necessary for initiating replication.

• Primer consists of ribonucleotides.

Leading and Lagging Strands

Leading Strand

• Synthesized continuously in the 5’ to 3’ direction using the 3’ to 5’ template strand.

Lagging Strand

• Template strand oriented 5’ to 3’, requiring synthesis in fragments (Okazaki fragments) since replication cannot occur in the 3’ to 5’ direction.

Okazaki Fragments

• Short, unconnected sequences formed on the lagging strand needing to be ligated together by DNA ligase.

RNA Primer Importance

• RNA primers are initially synthesized due to the difficulty of starting DNA strands.

• They allow for easy replacement and correction of errors later in replication.

Sliding Clamp

• Stabilizes and enhances the activity of DNA polymerase during synthesis.

Topoisomerases

Topoisomerase I

• Relieves tension in the DNA strand by creating a single-strand break, allowing rotational movement to relieve strain.

Topoisomerase II

• Functions by creating double-strand breaks, allowing another part of the helix to pass through, subsequently resealing the break.

DNA Replication Machine Components

• Various proteins including DNA helicase, DNA primase, DNA polymerases, and SSBs collaborate in the replication fork to synthesize new DNA.

Maintenance of DNA Sequences

• Changes in DNA sequences (mutations) can occasionally benefit species but also pose risks to individual survival.

• Mutation Rate: Typically ~1 base per 10^9 nucleotides per replication.

Proofreading Mechanisms

• Errors during replication are corrected by mechanisms before they become permanent mutations.

• DNA Polymerase Proofreading: Ensures base pair fidelity through conformational changes and stalling if a mismatch occurs.

• Exonucleolytic Proofreading: Processes any incorrectly paired bases, allowing polymerase to remove and replace them.

Tautomeric Forms

• Rare forms of bases can lead to mispairing during replication, potentially causing mistakes that are corrected through proofreading.

Packaging of New DNA

• Eukaryotic chromosomes have proteins (histones) that need to be synthesized and incorporated as DNA is replicated.

• Histone Modifications: Copied during replication, maintained by reader-writer complexes.

Telomere Replication

Challenges

• Lagging strand cannot synthesize at the end of the DNA molecule due to lack of binding site.

Telomerase

• Enzyme that adds repetitive nucleotide sequences to the ends of chromosomes (telomeres) using a built-in RNA template to extend the parental strand.

Telomere Structure and Regulation

• Longer 3' DNA strand looks like damaged DNA to repair machinery; it folds over to avoid detection.

• Telomere length is regulated, and telomerase activity can be diminished leading to cellular aging or death.

• Certain cancers are associated with uncontrolled telomerase activity.