Maintenance and expression of genetic information Central Dogma: DNA RNA Protein

GAATTGCGCCTTTTG

5’-GAATTGCGCCTTTTG-3 3’-CTTAACGCGGAAAAC-5’

Minor Groove Major Groove

DNA can be supercoiled

Semi-conservative Replication of DNA The Watson-Crick Model

Proof of the Watson-Crick Model: The Meselson-Stahl Experiment

# generations and 1.9 mixed 0 and 4.1 mixed

Starting DNA Heavy/Heavy 1st generation All Heavy/Light 2nd generation Two Heavy/Light Two Light/Light 3rd generation Two Heavy/Light Six Light/Light The Meselson-Stahl Experiment

DNA Polymerase

A 3’ hydroxyl group is necessary for addition of nucleotides

1’ 2’ 3’ 1’ 2’ 3’ 4’ 5’ 4’ 5’ 2’ 3’ 2’ 3’ 4’ 5’ 4’ 5’ 1’ DNA chain growth is driven by PPi release/hydrolysis

Accuracy of DNA polymerases is essential. --Error rate is less than 1 in Due in part to “reading” of complementary bases --also contains its own proofreading activity

DNA Polymerase contains a Proofreading subunit

Proofreading by DNA polymerase

Proofreading by DNA polymerase

Both Template strands are copied at a Replication Fork

The polarity of DNA synthesis creates an asymmetry between the leading strand and the lagging strand at the replication fork

Topoisomerase Protein complexes of the replication fork

Protein complexes of the replication fork: DNA polymerase DNA primase DNA Helicase ssDNA binding protein Sliding Clamp Clamp Loader DNA Ligase DNA Topoisomerase

DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging strand

Replication of the Lagging Strand

DNA ligase seals nicks left by lagging strand replication

DNA helicase unwinds the DNA duplex ahead of DNA polymerase creating single stranded DNA that can be used as a template

DNA helicase moves along one strand of the DNA

ssDNA binding proteins are required to “iron out” the unwound DNA

ssDNA binding proteins bind to the sugar phosphate backbone leaving the bases exposed for DNA polymerase

DNA polymerase is not very processive (ie it falls off the DNA easily). A “sliding clamp” is required to keep DNA polymerase on and allow duplication of long stretches of DNA

A “clamp loader:” complex is required to get the clamp onto the DNA

Lagging strand synthesis

MCM proteins PCNA RPC Topoisomerase

Ahead of the replication fork the DNA becomes supercoiled

The supercoiling ahead of the fork needs to be relieved or tension would build up (like coiling as spring) and block fork progression.

Supercoiling is relieved by the action of Topoisomerases. Type I topoisomerases: Make nicks in one DNA strands Can relieve supercoiling Type II topoisomersases Make nicks in both DNA strands (double strand break) Can relieve supercoiling and untangle linked DNA helices Both types of enzyme form covalent intermediates with the DNA

Topoisomerase I Action

Topoisomerase I Action

Topoisomerase II Action

Topoisomerase II Action

Topoisomerases as drug targets: Because dividing cells require greater topoisomerase activity due to increased DNA synthesis, topoisomerase inhibitors are used as chemotherapeutic agents. E.g. Camptothecin -- Topo I inhibitor Doxorubicin -- Topo II inhibitor These drugs act by stablilzing the DNA-Topoisomerase complex. Also, some antibiotics are inhibitors of the bacterial-specific toposisomerase DNA gyrase e.g. ciprofloxacin

DNA is replicated during S phase of the Cell Cycle

In S phase, DNA replication begins at origins of replication that are spread out across the chromosome

Each origin of replicaton Initiates the formation of bidirectional replication forks

Origins or replication are strictly controlled so that they “fire” only once per cell cycle Errors lead to overreplication of specific chromosomal regions. (= gene amplification) This seen commonly in cancer cells and can be an important prognostic indicator. It can also contribute to acquired drug resistance. E.g. Methotrexate induces amplification of the Dihydrofolate Reductase locus.

Errors of DNA Replication and Disease The rate of misincorporation of bases by DNA polymerase is extremely low, however repeated sequences can cause problems. In particular, trinucleotide repeats cause difficulties which can lead to expansion of these sequences. Depending where the repeat is located expansion of the sequence can have severe effects on the expression of a gene or the function of a protein.

Several mechanisms for the expansion of trinucleotide repeats have been proposed, but the precise mechanism is unknown. From Stryer: Looping out of repeats before replication.

Several inherited diseases are associated with expansion of trinucleotide repeat sequences. Very different disorders, but they share the characteristic of becoming more severe in succeeding generations due to progressive expansion of the repeats