Understanding DNA replication – and the resulting transmission of genetic information from cell to cell, and generation to generation – lays the groundwork for understanding the principles of heredity

Understanding DNA structure and replication is a prerequisite for understanding some of the principal tools of molecular biology

I. DNA structure: Three features of DNA makes it an ideal genetic material

A. Faithful replication

B. Information content

C. Capable of change

II. The building blocks of DNA

A. Nucleotides _____________________

B. Polynucleotides _____________________

C. Complementary base pairing _____________________

III. DNA replication is semiconservative : _______________________

IV. Requirements for DNA synthesis 

• dATP, dGTP, dTTP, and dCTP
• template DNA (a pre-existing single strand)
• DNA polymerase
  • There are multiple forms of DNA polymerase (in E. coli these are called polI, II, and III and in eukaryotes a,b,d,e,g). The different polymerases have different activities – those with direct roles in replication are called replicases. Others have secondary roles in replication and/or repair synthesis. Polymerases catalyze the polymerization of deoxyribonucleotides into a DNA chain. They catalyze formation of a phosphodiester bond between the 3'-OH of the deoxyribose on the last nucleotide and the 5' phosphate of the dNTP precursor, with a release of two of the three phosphates from the dNTP.  Polymerase is bound to the DNA and moves along template strand as polynucleotide chain grows. Thus, the dNTP precursor is identified that can base pair with the nucleotide on the template DNA. The frequency of error is low, but errors can occur. Polymerases can have exonuclease activity (they can remove nucleotides from the 3' end of the chain). This is a proofreading mechanism. The presence of an unpaired nucleotide from the 3'OH end of the growing chain triggers exonuclease activity: the unpaired nucleotide is cleaved from the end of the growing chain by the polymerase.

 

V. The essentials of DNA synthesis: Point: replication is 5' to 3'

VI. Details of DNA replication  

• Replication begins at a fixed point, called the origin, and proceeds bidirectionally. There is one origin in E. coli, which has some unique properties. In a higher plant, there are thousands of origins. 
• The DNA helix needs to be opened up. This is accomplished by helicase enzymes, which break the hydrogen bonds holding the two strands of the helix together.
• The unwound DNA is stabilized by a protein (single strand binding protein (SSB)).
• Priming: DNA polymerases can extend a chain, but they cannot start one.  Accordingly, DNA synthesis start with a primer.  For example, in E. coli, the primase enzyme synthesizes a short piece of RNA complimentary to the DNA. The RNA primers are later removed by exonuclease activity of a polymerase and replaced with DNA.
• Leading and lagging strands: DNA polymerases synthesize new chains only from 5' to 3', yet the DNA molecule is antiparallel and DNA synthesis is semi-conservative. DNA synthesis is continuous on one strand (the leading strand) and discontinuous on the other strand (the lagging strand). The DNA on the lagging strand is thus formed in fragments, called Okazaki fragments.
• Editing: Some polymerases have 3'-5' exonuclease activity. This allows these enzymes to "search" for mismatched bases that were incorrectly added during polymerization and removes them. This proofreading function occurs at the 3' end of the growing strand and proceeds 3' 5'.
• Summary of DNA replication....and more on RNA primers

VII. Telomeres and telomerase

The replication problem at chromosome ends: __________________

The telomerase solution (Figure); slides from Centre College _______________________

Chromosomes are capped at the ends with repetitive DNA sequences - the telomeres. Due to considerations in DNA replication, each time the cell divides, the telomere is shortened.  Eventually, the chromosomes become "so frayed that the cell senesces". In some cells- eggs, sperm, and cancer cells - an enzyme known as telomerase allows for "reconstruction" of the telomere, thus prolonging cell life.   Thus, there is indeed a fine line between life (eggs and sperm) and death (cancer).

2009 Nobel Prize in Physiology/Medicine:
Understand what telomeres are, what telomerase is, and why this research merited a Nobel Prize.

 

Required reading: How telomeres solve the end-protection problem. Titia de Lange. Science. November 2009: Vol. 326, pp. 948 - 952. DOI: 10.1126/science.1170633. At least the first three paragraphs

Text:  Chapter 7