Mechanisms by which human DNA primase chooses to polymerize a nucleoside triphosphate

Abstract

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha then elongates during the initiation of all new DNA strands. Even though primase misincorporates NTPs at a relatively high frequency, this likely does not impact the final DNA product since the RNA primer is replaced with DNA. We used an extensive series of purine and pyrimidine analogues to provide further insights into the mechanism by which primase chooses whether or not to polymerize a NTP. Primase readily polymerized a size-expanded cytosine analogue, 1,3-diaza-2-oxophenothiazine NTP, across from a templating G but not across from A. The enzyme did not efficiently polymerize NTPs incapable of forming two Watson-Crick hydrogen bonds with the templating base with the exception of UTP opposite purine deoxyribonucleoside. Likewise, primase did not generate base pairs between two nucleotides with altered Watson-Crick hydrogen-bonding patterns. Examining the mechanism of NTP polymerization revealed that human primase can misincorporate NTPs via both template misreading and a primer-template slippage mechanism. Together, these data demonstrate that human primase strongly depends on Watson-Crick hydrogen bonds for efficient nucleotide polymerization, much more so than the mechanistically related herpes primase, and provide insights into the potential roles of primer-template stability and base tautomerization during misincorporation.

Scheme 1

Misicorporation due to primer-template slippage.

Figure 1

Base analogues used in these studies.

Figure 2

Incorporation of NTP (analogues). Experiments contain 400 nM human primase, 1 mM NTP, and 2 μM 5’-[³²P]-DNAX. Products are labeled. Panels A-C represent incorporation of various NTP opposite a templating cytidine (panel A), hypoxanthine (panel B), and 2-pyridone (panel C). Panel A. In DNAX, X=C. Lane 1) no enzyme, no NTP; 2) no NTP; 3) GTP; 4) GTP and UTP; 5) UTP; 6) CTP; 7) ATP. Panel B. In DNAX, X=hypoxanthine. Lane 1) iso-GTP 2) benzimidazole NTP; 3) 2-pyridone NTP; 4) 2(1H)-pyrimidinone NTP; 5) 1-deazapurine NTP; 6) GTP; 7) ATP; 8) UTP; 9) CTP. Panel C. In DNAX, X=2-pyridone. Lane 1) iso-GTP 2) benzimidazole NTP; 3) 2-pyridone NTP; 4) 2(1H)-pyrimidinone NTP; 5) 1-deazapurine NTP; 6) GTP; 7) ATP; 8) UTP; 9) CTP. Panel D. DNAX, X=hypoxanthine. Assays contained the noted concentration of CTP (μM).

Figure 3

Efficient incorporation of UTP opposite purine but not purine NTP opposite T. Experiments contain 400 nM human primase, NTP, and 2 μM 5’-[³²P]-DNAX. Products are labeled Panel A. In DNAX, X=purine. The first lane contained no enzyme. Other lanes contained primase and the noted concentration of UTP (μM). Panel B. In DNAX, X=T. Assays contained the noted concentration of purine NTP (0 – 2000 μM).

Figure 4

Tautomerization of tC.

Figure 5

Incorporation of tCTP. Panel A. Assays contained 2 μM [³²P]DNAX, X=G. The first lane contained no enzyme. Other lanes contained 400 nM primase and the noted concentration of tCTP (0 – 2000 μM). Panel B. Assays contained 2 μM [³²P]DNAX, X=A. The first lane contained no enzyme., while latter lanes contained 400 nM primase and the noted concentration of tCTP (0 – 2000 μM). Panel C. Assays contained 2 μM [³²P]DNAX, X=G. The first lane contained no enzyme, while the other lanes contained primase, 800 μM of CTP and the noted concentration of tCTP (0 – 2000 μM).