C2005/F2401 '09 -- Recitation Problems #7 -- Hints & Answers

Hints:

1. A How does a DNA chain get started?

    B. What reaction does each of those enzymes catalyze? Is the corresponding function needed, and if so, what for?

2. A. Are the two strands parallel or not? Note that the top strand has its 3' on the left, not its 5'.

    B-1. Which way does the new strand grow? from it's 3' or 5' end?

    B-2. Are you making RNA or DNA?

    B-3. How is the sense strand related to the mRNA?

    C. Which is the lagging strand? The one that is synthesized continuously or discontinuously? Remember all chains must grow from their 5' ends toward their 3' ends, and each new chain must be antiparallel to the template.

    D-1. Where will each primer hybridize, if at all? When the primer is extended, which way will the chain grow?

    D-2. When do you need discontinuous synthesis, and when can you avoid it?

    E. How many strands are used as template for replication? Transcription?

Answers

1. A. U could be at the 5' end.

    B. Synthesis should involve primase, pyrophosphatase, & DNA polymerase.

Explanation: An Okazaki fragment is a discontinuously made section of the lagging strand at a DNA replication fork. It is involved in DNA synthesis, but synthesis of an Okaziki fragment starts with an RNA primer. You need primer (& primase) to start the fragment, because DNA polymerase can not start strands; it can only add to pre-existing ones. The primer is made of RNA -- that's why you can have a U in it. The U has to be at or near the 5' end, because the rest of the fragment is made of deoxyXTP's and has T, not U. The enzyme primase, which is a special type of RNA polymerase different from regular RNA polymerase, is needed to make the primer. Then DNA polymerase can add to the 3' end of the primer, elongating it. You don't need ligase to make a single Okazaki fragment; you only need ligase to shown individual fragments into a complete strand. Pyrophosphatase is needed to ensure that polynucleotide chain growth is energetically favorable, that is, that the overall ΔG of polymerization is very negative.

2. A. The DNA should look like this:      

    B-1. The top strand. The new chain must grow 5' to 3 and left to right, antiparallel to the template, so template must be the top strand.

    B-2. U will be on the 5' end.  The product must contain U because it is RNA.

    B-3. The bottom strand. The sense strand is the one that is not the template, and is the same as the mRNA, except that it has T instead of U.

    C-1. The bottom strand. Picture should describe or show one replication fork, not 2 bidirectional forks. Molecule should be a Y shape, with Y horizontal, and opening of Y on the left. New (leading) strand is made continuously, complementary to the top template strand; it grows 5' to 3', antiparallel to the template, left to right. New (lagging) strand is made discontinuously, complementary to the bottom template strand.

    C-2. CTGGA. Template strand on bottom contains sequence 5' TCCAG. New complementary lagging strand will contain the sequence 3' AGGTC 5' = CTGGA. (This sequence will be on the 3' end of the Okazaki fragment, not at the beginning, 5' end, which would include RNA primer.)

    C-3. The first Okazaki fragment.   Each segment (Okazaki fragment) of the lagging strand is made right to left & left-most fragment is made first.
   
    D-1. Neither.  The primer 5' AGGTC 3' would not hybridize to any of the sequences shown. It is parallel, not antiparallel, to the left end of the bottom strand. The primer 3' AGGTC 5' would hybridize to the left end of the bottom strand, but it could not act as primer to copy the DNA shown, because chain growth, if any, would occur in the wrong direction. Polymerase always adds to the 3' end of the primer, which is on the left of 3' AGGTC 5'.  There is no DNA here to act as template for growth to the left, and no 3' end to build on for growth of primer to the right.

    D-2. Neither strand.  All synthesis in PCR is continuous. Both strands are completely separated before replication, and the complement to each strand is made separately.

    E. Transcription should be different; replication should be the same.
    During DNA replication, both strands serve as template, and the entire molecule is replicated. The direction of the fork determines which new strand is made continuously and which is made discontinuously, but the end result is the same, no matter which direction the fork starts from.
    During transcription of any section of the DNA, only one strand serves as template. The newly made RNA strand must be antiparallel to the template (transcribed) strand, and the new RNA must grow 5' to 3'. Therefore the direction of transcription determines which strand of the DNA can serve as template. If you reverse the direction of transcription, you reverse which strand can be template.  Note that the terms "leading" and "lagging" strands apply only to replication, not to transcription.