Molecular Basis for Relationship between Genotype and Phenotype

1. Molecular Basis for Relationship between Genotype and Phenotype genotype DNA DNA sequence transcription replication RNA translation amino acid sequence protein function phenotype organism

2. Overview of DNA Synthesis DNA polymerases synthesize new strands in 5’ to 3’ direction. Primase makes RNA primer. Lagging strand DNA consists of Okazaki fragments. In E. coli, pol I fills in gaps in the lagging strand and removes RNA primer. Fragments are joined by DNA ligase.

3. DNA Replication at Growing Fork DNA polymerases add nucleotides in 5’ to 3’ direction. Because of antiparallel nature, synthesis of DNA is continuous for one strand and discontinuous for the other strand.

4. DNA Replication: Synthesis of Lagging Strand Several components and steps are involved in the discontinuous synthesis of the lagging strand. Note that DNA polymerases move in 3’ to 5’ direction on the template DNA sequence.

5. DNA Replication: Synthesis of Lagging Strand DNA extended from primers are called Okazakifragments. In E. coli, pol I removes RNA primers and fills in the gaps left in lagging strands. DNA ligase joins these pieces.

6. Replisome and Accessory Proteins Looping of template DNA for the lagging strand allows the two new strands to be synthesized by one dimer. pol III holoenzyme is a complex of many different proteins. Refer to Figure 7-20 from Introduction to Genetic Analysis, Griffiths etal., 2012.

7. Priming DNA Synthesis DNA polymerases can extend (but cannot start) a chain. Primase enzyme makes short RNA primer sequence complementary to template DNA. DNA polymerase extends RNA primer with DNA. Primosome is a set of proteins that are involved in the synthesis of RNA primers. Refer to Figure 7-20 from Introduction to Genetic Analysis, Griffiths etal., 2012.

8. Supercoiling results from separation of template strands during DNA replication.

9. Helicases and Topoisomerases Helicase enzymes disrupt hydrogen bonding between complementary bases. Single-stranded binding protein stabilizes unwound DNA. Unwound condition increases twisting and coiling, which can be relaxed by topoisomerases (such as DNA gyrase). Topoisomerases can either create or relax supercoiling. They can also induce or remove knots.

10. Chromatin assembly factor I (CAF-I) and histones are delivered to the replication fork. CAF-I and histones bind to proliferating cell nuclear antigen (PCNA), the eukaryotic version of clamp protein. Nucleosome assembly follows thereafter. Refer to Figure 7-23 from Introduction to Genetic Analysis, Griffiths etal., 2012.

11. Overview of DNA Synthesis DNA polymerases synthesize new strands in 5’ to 3’ direction. Primase makes RNA primer. Lagging strand DNA consists of Okazaki fragments. In E. coli, pol I fills in gaps in the lagging strand and removes RNA primer. Fragments are joined by DNA ligase.

13. Initiation at Origin of Replication Prokaryotes: Fixed origin DnaA proteins DnaB (helicase) Eukaryotes: Multiple origins ORC protein complex Cdc6 and Cdt1 MCM complex (helicase)