Computational Biology I LSM5191

1. Computational Biology ILSM5191 Aylwin Ng, D.Phil Lecture 1: Introduction to Nucleic Acids – the building blocks of life.

2. DNA & CHROMOSOMES 2m of DNA, all 3 billion letters in the DNA code, compacted into 46 chromosomes, and packed into a cell 0.0001cm across!

4. DNA densely packed into Chromosomes

5. Flow of Information in Living Systems Central Dogma of molecular biology: transcription translation DNA RNA  Protein DNA Sequence Implies Structure Implies Function

6. Transcription mRNA Transport Translation Nascent polypeptide mRNA ribosome Post-transl. modif functional protein

7. NUCLEIC ACIDS • Deoxyribonucleic acid (DNA) contains the information prescribing the amino acid sequence of proteins. • This information is arranged in units termed genes. • A GENE is the entire nucleic acid sequence that is necessary for the synthesis of a functional polypeptide • Ribonucleic acid (RNA) serves in the cellular machinery that chooses and links amino acids in the correct sequence. • DNA and RNA are polymers of nucleotide subunits

8. Nucleotide Phosphate group Base Ribose or Deoxyribose (shown here) NUCLEOTIDE SUBUNITS • A nucleotide unit consists of a pentose sugar, a phosphate moiety (containing up to 3 phosphate groups) and a Base. • Subunits are linked together by phosphodiester bond, to form a ‘sugar-phosphate backbone’:

9. NUCLEOTIDES • All nucleotides have a common structure

10. BASES • 5 principal bases in nucleic acids: A, G, C, T are present in DNA A, G, C, U are present in RNA

11. NUCLEOSIDES & NUCLEOTIDES

12. ELUCIDATING THE STRUCTURE OF DNA • James Watson (Cambridge University), • Francis Crick (Cambridge University), • Maurice Wilkins (King’s College London), • Rosalind Franklin (King’s College London) • - succeeded in obtaining superior X-ray diffraction data Nobel Prize (Medicine) in 1962

13. X-RAY DIFFRACTION • Data showed that DNA has the form of a regular helix • Diameter 20 Å (2 nm) • Making a complete turn every 34 Å (3.4 nm) •  i.e. 10 nucleotides per turn

14. BASE-PAIRING Edwin Chargaff’s results (1952): Base compositions experimentally determined for a variety of organisms

15. DNA STRUCTURE • Native DNA (B-form) is a double helix of complementary anti-parallel chains. • Double helix is right-handed, with turns running clockwise along helical axis. Hydrogen bonding between complementary base pairs (A-T or G-C) holds the two strands together

16. DNA REPLICATION • “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” • Watson & Crick, Nature (1953)

17. DNA REPLICATION • DNA replication is semi-conservative. • IMPLICATION: • the structure of DNA carries information needed to perpetuate its sequence . • Demonstrated by Meselson-Stahl (1958) • Labeled parental DNA with ‘heavy’ density label by growing E. coli in medium containing isotope (e.g. 15N): Light (14N) Hybrid Heavy (15N) parental 1st Gen 2nd Gen

18. NUCLEIC ACID SYNTHESIS • Both DNA and RNA chains are produced by copying of template DNA strands. • Nucleic acid strands grow in the 5’  3’ direction. • Energetically unfavorable. Driven by energy available in the triphosphates. • DNA-dependent RNA polymerases can initiate strand growth but DNA polymerases require a primer strand.

19. E. coli DNA polymerases Main replicating enzyme DNA repair DNA repair & replication

20. DNA Replication Clip http://academy.d20.co.edu/kadets/lundberg/DNA_animations/DNAreplication.mov

21. BIDIRECTIONAL REPLICATION • DNA replication proceeds bidirectionally from a given starting site (Origin of Replication), with both strands being copied at each fork. • Common features of Replication Origins (of E. coli, yeast, SV40) • Unique segments containing multiple short repeated sequences, • Short repeated units recognised by multimeric proteins (which assembles DNA polymerases & replication enzymes), • Origin regions contain an AT-rich stretch (less energy req.d to melt A.T base pairs).

22. BIDIRECTIONAL REPLICATION • Key events prior to the replication process (E. coli): • Binding of DnaA protein at Origin  separate (‘melt’) the strands. • DnaC & DnaB bind at Origin. • Then Helicase (DnaB)  unwinding of duplex in opposite directions away from Origin. • Unwinding of duplex is an ATP-dependent process. • Single-strand binding (SSB) protein binds to the single-stranded (ss) DNA, preventing it from reforming the duplex state. • Primases (RNA polymerase) bind to DnaB helicase  primosome complex • Primases dissociate after synthesizing short primer RNAs (complementary to both strands).

23. REPLICATION FORK

24. LAGGING-STRAND SYNTHESIS

25. TWO or just ONE Polymerase needed?

26. MAMMALIAN DNA POLYMERASES Main replicating enzyme Priming DNA repair Mitochond. DNA replication

27. REPLICATION IN EUKARYOTES • Very similar to replication in bacteria, differing only in details. • DNA polymerase  has primase activity  generates RNA primers. • DNA polymerase  is the main replicating enzyme. • Eukaryotic DNA polymerases appear to lack 5’ 3’ exonuclease activity needed to remove RNA primer from each Okazaki fragment. • ‘Flap endonuclease’ (FEN1) initiates primer degradation by associating with DNA polymerase .

28. What happens at Telomeres? Leading strand Chromosome end (Telomere) 3’ 5’ 5’ Parent molecule 3’ 3’ 5’ Lagging strand 3’ 5’ 5’ 3’ 2 Daughter molecules 200bp 200bp 200bp 200bp 200bp 3’ 5’ Missing Okazaki fragment 5’ 3’ 5’ Next Generation (or Grand-Daughter) molecule 3’ 5’ 3’ Molecule has become shorter

29. The Solution: TELOMERASE • Telomeres can be extended by an independent mechanism. • Catalysed by TELOMERASE. • Enzyme consists of both protein & RNA. • RNA is 450 nucleotides long. • Contains the seq. 5’-CUAACCCUAAC-3’ near its 5’ end. • Underlined seq. is the reverse complement of the human telomere repeat seq. 5’-TTAGGG-3’. • This allows telomerase to extend the 3’end a sufficient amount, • to facilitate priming & synthesis of a new Okazaki fragment by DNA polymerase  •  generate a double-stranded end.

30. How TELOMERASE extends 3’-end