Biochemistry Lectures

DNA replication recombination and repair, RNA synthesis and splicing

Reading material: Stryer, Chapters 27 and 28

Abstract: DNA is built as an antiparallel double helix with the stands bound together with hydrogen bonds between bases. Before cells divide, DNA molecules are copied to identical molecules in a process called replication. The replication is catalysed by the enzyme DNA polymerase. DNA replication starts at an origin and needs a DNA template (a DNA strand), deoxynucleotides, Mg2+ and a primer chain with a 3'-OH group. Replicataion is semiconservative, which means that one strand is newly synthesized whereas the other is passed on unchanged from the parent DNA molecule.

To separate the two strands of DNA, a helicase breaks the hydrogen bonds between the two strands. All synthesis takes place in the 5' 3' direction. The leading strand is synthesized continuously by DNA polymerase III (in E. coli). The lagging strand is synthesized discontinuously in Okazaki fragments by DNA polymerase III. The primers are synthesized by primase (a RNA polymerase). The primers in the lagging strand are removed by DNA polymerase I and the gaps are filled by this polymerase. The free ends of the DNA strand are joined by DNA ligase. The DNA polymerase III is highly processive and the sliding DNA clamp, formed by the beta subunits, is critical for processivity. Topoisomerases are required to regulate the twisting of the DNA. A number of mutations occur in DNA which are continually repaired by enzymes.

Specific parts of the DNA are copied to RNA transcripts, mRNA, which are used for the protein synthesis. Genes are transcribed to mRNA molecules by RNA polymerases. In prokaryotes, there are specific DNA sequences, promoters, that are recognized by RNA polymerase. The RNA polymerase makes a complementary copy of one of the DNA strands until a terminator site is reached.

Key concepts:
Nucleotides
4 bases
Ribose
Deoxyribose
Phosphodiester bonds
5' and 3' OH
DNA structure
Sugar-phosphate backbone
Double helix
Anti parallel
Charged groups outwards
Hydrophobic bases inward
Base pairing in normal B-DNA
Melting of DNA
Annealing
Replication
DNA polymerase I
DNA polymerase III holoenzyme
Proofreading
Nuclease domains
Leading strand
Lagging strand
Okazaki fragments
Helicase
Primase, primer
DNA ligase
Topoisomerases, gyrase
Mutations
DNA repair enzyme

RNA structure (single stranded, has secondary structure)
Differences between DNA/RNA, (deoxyribose/ribose, thymine/uracil)
Different types of RNA: tRNA, mRNA, rRNA
Transcription
RNA polymerase
Promoter

KE7001 Biochemistry Lectures